Rationale: Growing evidence indicates that oxidative stress contributes markedly to endothelial dysfunction. The selenoenzyme glutathione peroxidase 4 (Gpx4) is an intracellular antioxidant enzyme important for the protection of membranes by its unique activity to reduce complex hydroperoxides in membrane bilayers and lipoprotein particles. Yet a role of Gpx4 in endothelial cell function has remained enigmatic. Objective: To investigate the role of Gpx4 ablation and subsequent lipid peroxidation in the vascular compartment in vivo. Methods and Results: Endothelial-specific deletion of Gpx4 had no obvious impact on normal vascular homeostasis nor did it impair tumor-derived angiogenesis in mice maintained on a normal diet. By stark contrast, aortic explants from endothelial-specific Gpx4 knockout mice showed a markedly reduced number of endothelial branches in sprouting assays. To shed light onto this apparent discrepancy between the in vivo and ex vivo results, we depleted mice of a second antioxidant, vitamin E, which is normally absent under ex vivo conditions. Mice were therefore fed a vitamin E-depleted diet for 6 weeks before endothelial deletion of Gpx4 was induced by 4-hydroxytamoxifen. Surprisingly, about 80% of the knockout mice died. Histopathological analysis revealed detachment of endothelial cells from the basement membrane as well as endothelial cell death in multiple organs which triggered thrombus formation. Thromboembolic events were the likely cause of various clinical pathologies including heart failure, renal and splenic micro-infarctions or paraplegia. Conclusions: Here we show for the first time that in the absence of Gpx4, sufficient vitamin E supplementation is crucial for endothelial viability.