Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy. Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. In this study, we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro. Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53, resulting in a pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo. p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA. These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.