Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for the treatment of cancer because it targets tumor cells while sparing normal cells. Several cancers, however, fail to respond to TRAIL's antineoplastic effects. These resistant tumors require cotreatment with sensitizing agents in order for TRAIL to exert anticancer activity. Histone deacetylase inhibitors (HDACi) have been recognized as potent TRAIL sensitizers. In searching for the determinants of TRAIL responsiveness, HDACi-mediated TRAIL sensitization has been predominantly attributed to TRAIL receptor upregulation. This explanation, however, has been challenged by a few studies. The aim of the present study was to explore the relevance of TRAIL receptor expression for HDACi-mediated TRAIL sensitization in childhood tumors, i.e., in medulloblastoma, Ewing's sarcoma and osteosarcoma. In previous studies, we had shown that TRAIL and HDACi were synergistic in inducing apoptosis in medulloblastoma and Ewing's sarcoma. In the present study, we demonstrate that HDACi cooperated with TRAIL in eliciting cell death in osteosarcoma. However, HDACi treatment did not alter or even reduced cell surface expression of TRAIL receptors in the three childhood tumors. In gaining insight into the apoptotic pathway involved in TRAIL sensitization, HDACi were found to potentiate TRAIL-induced caspase-8 activation. Taken together, our findings suggest that HDACi-mediated TRAIL sensitization is not the result of TRAIL receptor upregulation, but the result of a receptor-proximal event in childhood tumor cells.