Purpose: Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear. Experimental Design: To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo. Results: CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38α-/- cells or an inhibitor of p38, we show that the presence and activation of p38α is important for cytotoxicity in all cell types examined. Conclusions: Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.