Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4^−/− embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4^+/− mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4^+/− mice are markedly sensitive to inducers of oxidative stress, including γ-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4^+/− mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.