Magdalene, or Maggy, is unlocked when the player obtains seven or more heart containers at once in a run. Magdalene starts with four red heart containers and the Yum Heart. If the Aprils Fool challenge is completed, she will also start with a Speed Up pill.
Cain is unlocked when the player owns 55 or more coins at once in a run. He starts with two red heart containers, the Lucky Foot, and one key.
Cain will also start with the Paper Clip trinket if 68 coins have been donated to the Greed Machine.
Judas is a character unlocked after beating Satan for the first time. Judas starts with one red heart container but has The Book of Belial upon starting, as well as 3 coins.
???, also known as the Blue Baby, is a character unlocked by defeating Mom's Heart ten times. He starts with three soul hearts, and his health meter is unique in that he cannot gain red heart containers. If an item would grant him a red heart container (including an empty one), it adds a soul heart instead.
Eve is a character unlocked by beating two floors in a row without collecting any heart pickups. Eve starts with two red heart containers, Whore of Babylon, and the Dead Bird.
Eve will also start with the Razor Blade upon donating 439 coins to the Greed Machine.
However, if the Razor Blade has not been unlocked by defeating Satan with Eve, Eve will not start with the Razor Blade until both conditions have been fulfilled.
This was changed in AB+ Booster Pack 5. Eve will start with the Razor Blade regardless of whether it is unlocked or not as long as 439 coins have been donated to the Greed Machine.
Samson is unlocked when the player defeats two floors in a row without taking any damage. Samson starts with 3 red heart containers and Bloody Lust. Every time Samson is dealt damage, he receives a damage boost that resets when he goes down a floor.
Samson will start with the Child's Heart trinket after completing the Ultra Hard challenge.
Azazel is a character in The Binding of Isaac: Rebirth unlocked by making three deals with the devil in one run. Azazel starts with a short-ranged version of Brimstone, the ability to fly, the card 0, The Fool, and three black hearts without any regular heart containers; however, unlike ???, Azazel can still obtain heart containers with items.
Lazarus is unlocked once the player has four or more soul hearts (or black hearts) at once during a run. He is a new character in The Binding of Isaac: Rebirth. He starts with three red heart containers and a random pill. He has slightly lower Range and Luck than Isaac but also has 1 extra life at the beginning of the game.
His revived form has increased base stats, with above-average damage, speed, and range. Though low on health, his improved stats counterbalance this weakness well.
After Challenge 31: Backasswards is beaten, Lazarus will start with Anemic
Eden is a new character in The Binding of Isaac: Rebirth, unlocked by completing The Womb for the first time. Eden is unique in that they are only playable when the player has at least one Eden Token. Eden Tokens are awarded every time Mom's Heart, It Lives, or Ultra Greed is defeated. Each time a game is started as Eden, one token is consumed and a new Eden is randomly generated based on the seed, with one of the multiple hairstyles, random base stats, and two randomly chosen starting items.
Eden's stats are centered around Isaac's, meaning that they are on average identical to Isaac's but may vary significantly from run to run. The stats you see on this page are all ranges with Isaac's stats at the center, plus or minus a certain amount up to the values listed.
For details regarding the exact generation of an Eden Run, check Eden Generation.
The Lost is a secret character in The Binding of Isaac: Rebirth.
The Lost starts with no health and cannot gain health by any means. Therefore, it will die from any hit. The Lost starts with flight, Spectral tears, D4 (if unlocked) and Holy Mantle (after donating 879 coins to the Greed Machine). The Lost can take devil deals for free and activate the button in Greed mode without taking damage.
Lilith is a character added in The Binding of Isaac: Afterbirth expansion. Lilith starts with one red heart container and two black hearts. Lilith's starting items are the Box of Friends, Incubus and Cambion Conception. Lilith is permanently blindfolded, meaning she has no ability to fire tears; she instead deals damage with the Incubus familiar that follows her.
Lilith is unlocked by completing Greed mode with Azazel.
The Keeper is a secret character added in The Binding of Isaac: Afterbirth.
Keeper is unlocked by donating 1000 coins to the Greed machine. Keeper starts with a triple shot and 1 bomb, he starts with Wooden Nickel after defeating Isaac with Keeper, with 1 coin pickup after defeating Hush with Keeper, with Store Key after defeating Satan with Keeper. Unlike other characters, Keeper uses coins as health. He starts with two health coins, loses one every time he is damaged, and heals for one coin every time he picks up a coin. All types of hearts are transformed into blue flies. If Keeper falls to one health coin (Devil Deal, Health Down, etc.), any HP Up will increase his health back to two health coins.
Apollyon starts with two heart containers and Void, a 6-charge item that destroys items and grants bonuses in return and combines all voided active items into one. Apollyon is unlocked by defeating Mega Satan for the first time.
The Forgotten is a secret character added in Booster Pack #5 that resembles a Bony. He cannot fire regular tears, and instead has a bone club that can be swung as a melee weapon or charged to be thrown.
The Forgotten cannot acquire regular heart containers. Any regular heart containers acquired will be turned into bone hearts, and any soul or black hearts will be given to The Soul. The Forgotten and The Soul share a health bar, so each form can only reach a maximum of 6 hearts each instead of the regular 12.
When a character dies after picking up the item Judas' Shadow, they will respawn as Dark Judas. Dark Judas spawns with two black hearts, a damage multiplier of 2.00 (instead of Judas' 1.35 multiplier), and a base speed of 1.1. Similar to Azazel, Dark Judas can still gain heart containers despite starting with none. Dark Judas has the highest base damage multiplier of all the playable characters.