Tumors that express wild-type P53 provide a target for therapies designed to reactivate P53 function. This is supported by the potent activation of P53 in tumor cells by Nutlin, a cis-imidazoline that inhibits the Hdm2-P53 interaction. The efficacy of Hdm2.P53 antagonists could be compromised if they do not antagonize Hdmx, an Hdm2 homolog that inhibits P53 transactivation. We evaluated the role of Hdmx expression in sensitivity to Nutlin in a range of cancer cell lines. Nutlin reduced Hdmx levels in normal cells and some cancer cell lines, whereas other cancer cells were refractory to such down-regulation. Strikingly, Nutlin did not disrupt Hdmx.P53 complexes, and in cell lines where no Hdmx degradation occurred, Nutlin failed to induce apoptosis. shRNA-mediated reduction of Hdmx sensitized cells to apoptosis, but caspase-3 was neither required nor sufficient for Hdmx degradation or apoptosis. Our data imply that Hdmx is an important determinant of the outcome of P53 activation. Thus, targeting Hdmx may be a therapeutic strategy that complements drugs such as Nutlin.