Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56Lck-deficient cells to show that CD95-induced cell death is highly dependent on p56Lck activity and its localization within plasma membrane. We found that p56Lck acts upstream of the mitochondria; in the absence of p56Lck, Bid cleavage and the release of cytochrome c was severely impaired. Moreover, p56Lck-deficient cells or cells expressing inactive form of p56Lck displayed defective formation of the DISC post CD95 stimulation. In vivo reconstitution of thymocytes from p56lck deficient mice, which are resistant to apoptosis, with p56Lck restored Fas-mediated cell death. Our results support a novel model whereby sensitivity to apoptosis is regulated through quantitative changes in the stoichometry of DISC components triggered by p56Lck activation and localization.