The are multiple characters that can be unlocked in the game, each requiring you to fulfill a certain condition during a play-through. Note: You can only unlock characters in regular, random runs. Meeting the requirements in a challenge or manually inputting a random seed will not allow you to unlock characters (indicated by the no trophy icon below your stats).

Character	How to unlock
??? (The Blue Baby)	Reward for defeating Mom's Heart for the 10th time.
Azazel	Make three deals with the devil during a run.
Cain	Have 55 or more pennies at any time during a run.
Eden	Reward for defeating Mom's Heart for the first time.
Eve	Complete two consecutive floors without picking up any hearts (including Soul, Black, and Eternal Hearts)
Judas	Unlocked when you defeat Satan the first time.
Lazarus	Have 4 or more Soul and/or Black Hearts at any time during a run.
Magdalene	Have 7 or more Red Heart Containers at any time in a run
Samson	Complete two consecutive floors without taking any damage.