Nutlin-3, a human double minute 2 (HDM2) antagonist, induces cell cycle arrest or apoptosis by upregulating p53 in cancer cells. WT1, the product of Wilms' tumor gene 1, has been shown to interact with p53, but the effect of WT1 on nutlin-3-induced apoptosis has yet to be examined. To address this issue, we analyzed the inhibitory effect of nutlin-3 on cell growth as a function of Wt1 expression status using a Wt1-inducible U2OS cell line. In the absence of Wt1 expression, nutlin-3 induced cell cycle arrest with marginal cytotoxicity. Furthermore, upon Wt1 expression, nutlin-3 exerted a marked degree of cell death, as evidenced by the accumulation of hypo-diploid cells and LDH release. During cell death induction, cytochrome c was released into the cytosol, and caspase-9 and -3 were activated, suggesting that an intrinsic apoptotic pathway may be involved in this cell death. Consistent with this, z-VAD-Fmk, a pan-caspase inhibitor and the overexpression of BCL-XL attenuated the cell death. Nutlin-3 caused an increase in the mRNA levels of both BCL-XL and BAK, as well as their corresponding protein levels in mitochondria. In the presence of Wt1, nutlin-3-induced BCL-XL expression was attenuated while the expression of nutlin-3-induced BAK was potentiated. Collectively, these results suggest that WT1 potentiates nutlin-3-induced apoptosis by downregulating the expression of BCL-XL while upregulating that of BAK, which leads to the activation of an intrinsic apoptotic pathway.