In the Repentance expansion, two characters ( Bethany and Jacob and Esau) were added. In the Afterbirth † expansion, one character ( Apollyon) was added, plus one more secret character ( The Forgotten) added via a Booster Pack. In the Afterbirth expansion, one new character ( Lilith) was added, plus one more character via an update ( Keeper). Rebirth contains four new characters ( Azazel, Lazarus, Eden, and The Lost).
Eve was added in The Binding of Isaac's Halloween Update, while Samson was added in the Wrath of the Lamb expansion. Five of these characters ( Isaac, Magdalene, Cain, Judas, and ?) came with the original The Binding of Isaac, while two additional characters ( Eve and Samson) were added later. There are 34 playable characters in The Binding of Isaac: Rebirth, with 11 available in the base game and 23 available in the DLCs.