The death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), has shown great promise for inducing apoptosis selectively in tumors. Although many tumor cells are resistant to TRAIL-induced apoptosis alone, they can often be sensitized by co-treatment with DNA-damaging agents such as etoposide. However, the molecular mechanism underlying this therapeutically important synergy is unknown. We explored the mechanism mediating TRAIL-DNA damage apoptotic synergy in human mesothelioma cells, a tumor type particularly refractory to existing therapies. We show that Bid, a cytoplasmic Bcl-2 homology domain 3-containing protein activated by caspase 8 in response to TRAIL ligation, is essential for TRAIL-etoposide apo-ptotic synergy and, furthermore, that exposure to DNA damage primes cells to induction of apoptosis by otherwise sublethal levels of activated Bid. Finally, we show that the extensive caspase 8 cleavage seen during TRAIL-etoposide synergy is a consequence and not a cause of the apoptotic cascade activated downstream of Bid. These data indicate that TRAIL-etoposide apoptotic synergy arises because DNA damage increases the inherent sensitivity of cells to levels of TRAIL-activated Bid that would otherwise be insufficient for apoptosis. Such studies indicate how the adroit combination of differing proapoptotic and sublethal signals can provide an effective strategy for treating refractory tumors.