Apoptosis-related genes play important roles in thymocyte maturation. We show that cellular FLICE-like inhibitory protein (c-FLIP), a procaspase-8-like apoptotic regulator, plays an essential role in the efficient development of mature T lymphocytes. Mice conditionally lacking c-FLIP in T lymphocytes display severe defects in the development of mature T cells, as indicated by a dramatically reduced number of CD4+ and CD8+ T cells in the spleen and lymph nodes of mutant mice. The impaired T lymphocyte maturation in c-FLIP conditional knockout mice occurs at the single-positive thymocyte stage and may be caused by enhanced apoptosis in vivo. Moreover, although c-FLIP has been implicated in T cell receptor signaling through nuclear factor (NF)-kappaB and Erk pathways, activation of NF-kappaB and Erk in c-FLIP-deficient thymocytes appears largely intact. Collectively, our data suggest that the primary role of c-FLIP in thymocyte maturation is to protect cells from apoptosis.