The FLICE-inhibitory protein (FLIP) plays a key role in the regulation of apoptosis triggered by death ligands. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in some types of tumor but not in others. To assess the possible role of FLIP in apoptosis resistance in endometrial carcinoma, we performed an immunohistochemical study on a tissue microarray composed of 95 endometrial carcinomas. We found positive signals in 43% of the cases, as well as a significant difference in FLIP expression between stage I and II tumors. Moreover, we observed that endometrial carcinoma cell lines Ishikawa and KLE did not undergo apoptosis after TRAIL treatment. Cotreatment of these cells with the inhibitor of transcription actinomycin D resulted in a dramatic decrease in cell viability and induced activation of caspase-8. These events coincided with downregulation of FLIP mRNA and protein. Inhibitors of caspase-8 or overexpression of FLIP completely blocked apoptosis induced by actinomycin D plus TRAIL cotreatment. More importantly, downregulation of endogenous FLIP expression by specific siRNAs sensitized endometrial carcinoma cells to TRAIL-induced apoptosis in the absence of actinomycin D. Taken together, our results suggest for the first time a critical role for FLIP in the regulation apoptosis triggered by TRAIL in endometrial carcinoma cells.