Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasL(TET)) [Hyer et al., Molecular Therapy, 2000; 2:348-58 (ref.12)]. The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasL(TET) to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasL(TET) is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasL(TET) treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.