Granzyme B (GzmB) is a component of cytotoxic lymphocyte granules that can rapidly initiate apoptosis in target cells. While several procaspases are cleaved and activated by GzmB, the absolute requirement of caspase activation for GzmB-induced apoptosis is controversial. In this report, we demonstrate that GzmB can initiate apoptosis in the absence of caspase-3 activity by directly cleaving DFF45/ICAD to liberate activated DFF40/CAD. DFF45/ICAD cleavage occurs less efficiently in cells that lack caspase-3 activity, suggesting that the caspases normally amplify the GzmB death signal. DFF45/ICAD-deficient mouse embryo fibroblasts are partially resistant to GzmB-induced death, demonstrating the biological importance of DFF45/ICAD for GzmB-mediated apoptosis.