The effects of CD40 ligation in an epithelial context are complex, with the level of CD40 engagement influencing the physiological outcome. Low levels of CD40 ligation promote cell survival/proliferation, whereas high levels induce growth arrest/apoptosis. The precise form of the CD40 stimulus affects these responses with the most profound effects in carcinoma cells being induced by membrane-bound rather than recombinant soluble CD40L. However, the signaling pathways underlying these differential responses are yet to be fully characterized. We have investigated the mechanistic differences resulting from CD40 engagement by soluble and membrane-bound ligands using a novel adenovirus-delivered CD40L mutated to resist cleavage from the cell membrane in the CD40-positive EJ bladder carcinoma cell line. We have shown that membrane-bound CD40L induces apoptosis by influencing the balance between apoptotic and survival signals. Thus, membrane-bound CD40L stabilizes TNFR-associated factor 3 to induce JNK-dependent apoptosis via release of mitochondrial cytochrome c, caspase 9, and effector caspases 3/7. Further, we have shown that this process is dependent on activation of caspase 8. However, there is also a requirement for suppression of TNFR-associated factor 6-mediated PI3K/Akt-dependent survival signals for apoptosis to occur. These data provide mechanistic insights into the consequences of CD40 activation in carcinoma cells and how these might be exploited in the clinical development of CD40-targeted anticancer therapies.