Natural killer (NK) and CD1d-restricted Vα14i natural killer T (NKT) cells play a critical early role in host defense. Here we show that mice with a targeted deletion of T-bet, a T-box transcription factor required for Th1 cell differentiation, have a profound, stem cell-intrinsic defect in their ability to generate mature NK and Vα14i NKT cells. Both cell types fail to complete normal terminal maturation and are present in decreased numbers in peripheral lymphoid organs of T-bet-/- mice. T-bet expression is regulated during NK cell differentiation by NK-activating receptors and cytokines known to control NK development and effector function. Our results identify T-bet as a key factor in the terminal maturation and peripheral homeostasis of NK and Vα14i NKT cells.