The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lymphoblastic leukemia (ALL). We used a set of ALL cell lines with wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survivin expression in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3-resistant cells were either pretransfected with the PTEN gene or simultaneously treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfering RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3-resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapoptotic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL.