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The aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported to correlate with adverse clinical outcome in human glioblastoma in vivo. However, the question of how this survival network can be successfully targeted to restore the sensitivity of glioblastoma to apoptosis induction has not yet been answered. Here, we report that inhibition of PI3K by LY294002 broadly sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor- and chemotherapy-induced apoptosis, whereas mammalian target of rapamycin (mTOR) inhibition is not sufficient to restore apoptosis sensitivity. LY294002 significantly enhances apoptosis triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110alpha and/or p110beta by RNA interference (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. In contrast to PI3K inhibition, pharmacologic or genetic blockade of mTOR by RAD001 (everolimus), rapamycin, or RNAi fails to enhance TRAIL- or doxorubicin-induced apoptosis. Analysis of apoptosis pathways reveals that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis, which are abolished by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Most importantly, PI3K inhibition by LY294002 sensitizes primary cultured glioblastoma cells obtained from surgical specimens to TRAIL- or chemotherapy-induced cell death. By showing that PI3K inhibition broadly primes glioblastoma cells for apoptosis, our findings provide the rationale for using PI3K inhibitors in combination regimens to enhance TRAIL- or chemotherapy-induced apoptosis in glioblastoma.