Sensitivity to TRAIL-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and shRNA-targeted inhibition of cathepsin B reduced apoptosis. Because cFLIP inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced cFLIP_L expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP_L-overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP_L-overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-B activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells, and suggest cFLIP_L cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.