Current treatments for Glioblastoma multiforme (GBM) involve surgery, radiotherapy, and cytotoxic chemotherapy; however, these treatments are not effective and there is an urgent need for better treatments. We investigated GBM cell killing by a novel drug combination involving DT-EGF, an Epidermal Growth Factor Receptor-targeted bacterial toxin, and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) or antibodies that activate the TRAIL receptors DR4 and DR5. DT-EGF kills GBM cells by a non apoptotic mechanism whereas TRAIL kills by inducing apoptosis. GBM cells treated with DT-EGF and TRAIL were killed in a synergistic fashion in vitro and the combination was more effective than either treatment alone in vivo. Tumor cell death with the combination occurred by caspase activation and apoptosis due to DT-EGF positively regulating TRAIL killing by depleting FLIP, a selective inhibitor of TRAIL receptor-induced apoptosis. These data provide a mechanism-based rationale for combining targeted toxins and TRAIL receptor agonists to treat GBM.