Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II–null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non–cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.