Despite improvements in chemotherapy and surgery in the treatment of osteosarcoma, satisfactory results are still difficult to achieve. New therapeutic modalities need to be developed for the improvement of these treatments. TRAIL (TNF-related apoptosis inducing ligand) is known as a selective apoptosis inducer in most tumor cells, but not in normal cells. Therefore, TRAIL is a good candidate target for the treatment of tumors. However, sensitivity of osteosarcoma cells to TRAIL-induced apoptosis is lower than that of other types of tumor cells. Recently, DAP3 (death associated protein 3) was demonstrated to play a critical role in TRAIL-mediated apoptosis through activation of pro-caspase-8. Here, we found that LKB1, a serine/threonine kinase, expressed in bone and soft tissue sarcoma cells, associated with DAP3. We also demonstrated that expression of DAP3 induced apoptosis in osteosarcoma cells. Furthermore, expression of LKB1 induced apoptosis and co-expression of LKB1 with DAP3 strongly induced apoptosis in osteosarcoma cells. In addition, expression of LKB1 kinase dead mutant, LKB1 (K78M), inhibited DAP3-induced apoptosis in these cells. These results suggest that LKB1 is critical for TRAIL-induced apoptosis induction, cooperating with DAP3 in osteosarcoma cells. It is predicted that LKB1 and DAP3 could be critical target molecules for the treatment of osteosarcomas.