Notch/RBP-J signaling is required for generation of early T progenitors (ETP) and promotion of double-negative (DN) 4 cells from DN3 cells in thymocyte differentiation. However, whether Notch affects other steps during thymocyte differentiation remains unknown. Msx2-interacting nuclear target protein (Mint) is an endogenous inhibitor of Notch regulation. Concordantly, by ex vivo analyses of embryonic thymi and in vitro differentiation studies of fetal liver progenitors, we find that Mint deficiency enhances generation of ETP and DN4 cells. Unexpectedly, however, Mint deficiency impairs differentiation of ETP into DN2 cells, suggesting that Notch/RBP-J signaling negatively regulates DN1–DN2 transition.