To better understand the cytokine death-signal transduction pathways in human beta cells, we investigated the inhibitory effects of Bcl-2 (protooncogene bcl-2) and X-linked inhibitor of apoptosis (XIAP) on TRAIL (TNF-related apoptosis-inducing ligand)-induced human beta-cell destruction. A panel of Bcl-2-overexpressing transfectants of the human beta-cell lines NES2Y and CM was developed by transfection with a pEFpGKpuro vector containing Bcl-2 or an empty vector as a control. TRAIL-induced cytotoxicity and apoptosis of Bcl-2-overexpressing beta cells were clearly decreased, in comparison with wild-type cells and the empty vector transfectants. XIAP-overexpressing CM, NES2Y, and primary islet cells were generated by exposing cells to recombinant adenovirus-expressing XIAP (AdXIAP) or AdLacz as a control. TRAIL-induced cytotoxicity and apoptosis of CM, NES2Y, and primary islet cells infected with AdXIAP were clearly reduced compared with controls. Interestingly, cytotoxicity induced by TRAIL in human beta cells transfected with both Bcl-2 and AdXIAP was much less than that observed in human beta cells transfected with either Bcl-2 or XIAP alone (p < 0.005 in CM and p < 0.03 in NES2Y). Overexpression of both Bcl-2 and XIAP inhibited TRAIL-induced activation of caspases as well as TRAIL-mediated damage of mitochondrial function in cells, suggesting possible regulatory mechanisms. These results indicate that Bcl-2 and XIAP synergistically inhibit TRAIL-mediated death pathways in human beta cells.