In vitro studies suggest that Ras activation is necessary for erythroid cell development. However, genetic inactivation of the Ras isoforms H-Ras, N-Ras, and K-Ras in mice reportedly did not affect adult or fetal erythropoiesis, though K-Ras(-/-) embryos were anemic. Given these discrepancies, we performed a more detailed analysis of fetal erythropoiesis in K-Ras(-/-) embryos. Day-13.5 K-Ras(-/-) embryos were pale with a marked reduction of mature erythrocytes in their fetal livers. The frequency and number of both early (erythroid burst-forming unit [BFU-E]) and late erythroid progenitors (erythroid colony-forming unit [CFU-E]) were reduced in K-Ras(-/-) fetal livers compared with wild-type controls and displayed a delay in terminal erythroid cell maturation. Further, K-Ras(-/-) hematopoietic progenitors had reduced proliferation in response to erythropoietin and Kit ligand compared with control cells. Thus, these studies identify K-Ras as a unique Ras isoform that is essential for regulating fetal erythropoiesis in vivo.