Tyrosine kinases of the Src family have been implicated in key biological processes. Here, we provide evidence that p56 Lck, a lymphoid-specific Src kinase, is involved in the activation of the mitochondrial apoptosis pathway. Lck-deficient T cells were completely resistant to anticancer drugs. In contrast, apoptosis sensitivity to death receptors was not altered, indicating a specific interference of Lck with the mitochondrial pathway. Re-expression of Lck restored sensitivity to drug-induced apoptosis and triggered mitochondrial cytochrome c release and caspase activation. Further analysis identified that the sensitization by Lck was independent of classical mediators of T-cell signaling, but essentially involved the Bcl-2 protein Bak. Expression of Bak was completely absent in Lck-deficient cells, while re-expression of Lck transcriptionally triggered Bak expression and conferred sensitivity to apoptosis, associated with a proapoptotic conformational change of Bak. Furthermore, in vitro the truncated fragment of Bid specifically activated Bak and cytochrome c release only from mitochondria of Lck-expressing cells. These results do not only demonstrate a sentinel role of Lck in drug resistance but also delineate a hitherto unknown pathway of Src kinases in regulation of Bcl-2 proteins.