Abstract To potentiate the response of acute myelogenous leukemia (AML) cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) cytotoxicity, we have examined the efficacy of a combination with perifosine, a novel phosphatidylinositol-3-kinase (PI3K)/Akt signaling inhibitor. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease the cellular FLICE-inhibitory protein (cFLIP) in human lung cancer cell lines. Perifosine and TRAIL both induced cell death by apoptosis in the THP-1 AML cell line, which is characterized by constitutive PI3K/Akt activation, but lacks functional p53. Perifosine, at concentrations below IC50, dephosphorylated Akt and increased TRAIL-R2 levels, as shown by Western blot, reverse transcription-PCR, and flow cytometric analysis. Perifosine also decreased the long isoform of cFLIP (cFLIP-L) and the X-linked inhibitor of apoptosis protein (XIAP) expression. Perifosine and TRAIL synergized to activate caspase-8 and induce apoptosis, which was blocked by a caspase-8–selective inhibitor. Up-regulation of TRAIL-R2 expression was dependent on a protein kinase Cα/c-Jun-NH2-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine also synergized with TRAIL in primary AML cells displaying constitutive activation of the Akt pathway by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 up-regulation, cFLIP-L and XIAP down-regulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34+ cells from patients with AML. In contrast, CD34+ cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination of perifosine and TRAIL might offer a novel therapeutic strategy for AML. [Cancer Res 2008;68(22):9394–403]