RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of γδ T cells, whereas αβ T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4⁺ T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates γδ T cell generation and migration, αβ T cell maturation, terminal differentiation of CD4⁺ T cells into Th1/Th2 cells, and activation of T cells.