Activation-induced cell death (AICD) is involved in peripheral tolerance by controlling the expansion of repeatedly stimulated T cells via an apoptotic Fas (CD95; APO-1)-dependent pathway. The TNFRSF-6 gene encoding Fas is mutated in children suffering from autoimmune lymphoproliferative syndrome (ALPS), which is characterized by lymphoproliferation and autoimmunity. We examined AICD in Fas-deficient T cells from ALPS patients. We showed that primary activated Fas-deficient T cells die by apoptosis after repeated T cell antigen receptor (TCR) stimulation despite resistance to Fas-mediated cell death. This Fas-independent AICD was found to be mediated through a cytotoxic granules-dependent pathway. Cytotoxic granules-mediated AICD was also detected in normal T lymphocytes though to a lesser extent. As expected, the cytotoxic granules-dependent AICD was abolished in T cells from Rab27a- or perforin-deficient patients who exhibited defective granules-dependent cytotoxicity. Supporting an in vivo relevance of the cytotoxic granules-dependent AICD in ALPS patients, we detected an increased number of circulating T lymphocytes expressing granzymes A and B. Altogether, these data indicated that the cytotoxic granules-dependent cell death in ALPS may compensate for Fas deficiency in T lymphocytes. Furthermore, they identified a novel AICD pathway as a unique alternative to Fas apoptosis in human peripheral T lymphocytes.