Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.42) is an essential enzyme for the rapid production of NADPH, as required on exposure to oxidative stress. Mouse embryonic stem (ES) cells can produce all embryonic and fetal/adult cell types. By studying the in vitro differentiation of embryoid bodies produced from G6pdDelta ES cells that are totally unable to produce G6PD protein, we found that these cells are able to differentiate into mesodermal cells, cardiomyocytes, hepatocytes, and primitive erythroid cells. However, we show here that, after the hemoglobin switch has taken place, definitive erythrocytes die by apoptosis. This apoptotic death is delayed by reducing agents and by a caspase inhibitor, but it is prevented only by the restoration of G6PD activity. Thus, G6PD proves indispensable for definitive erythropoiesis.