Agents that generate reactive oxygen species (ROS) are recognized to enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24 (Ad.5/3-mda-7) caused plasma membrane clustering of CD95 and CD95 association with pro-caspase 8, effects that were enhanced by combined exposure to 17-N-allylamino-17-demethoxygeldanamycin (17AAG), As2O 3, or fenretinide and that correlated with enhanced cell killing. Knockdown of CD95 or expression of cellular FADD (Fas-associated protein with death domain)-like interleukin-1β-converting enzyme inhibitory protein, short form (c-FLIP-s) blocked enhanced killing. Inhibition of ROS generation, elevated cytosolic Ca2+, or de novo ceramide synthesis blocked Ad.5/3-mda-7 ± agent-induced CD95 activation and the enhancement of apoptosis. Ad.5/3-mda-7 increased ceramide levels in a PERK-dependent fashion that were responsible for elevated cytosolic Ca2+ levels that promoted ROS generation; 17AAG did not further enhance cytokine-induced ceramide generation. In vivo, infection of RCC tumors with Ad.5/3-mda-7 suppressed the growth of infected tumors that was enhanced by exposure to 17AAG. Our data indicate that in RCCs, Ad.5/3-mda-7-induced ceramide generation plays a central role in tumor cell killing and inhibition of multiple signaling pathways may have utility in promoting MDA-7/IL-24 lethality in renal cancer.