Although erythropoietin (EPO) and its receptor (EPOR) are crucial for the proliferation, survival, and terminal differentiation of erythroid progenitors, it remains to be elucidated whether EPOR-unique signaling is required for erythropoiesis. To address this issue, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR)-transgenic mice and heterozygous EPOR mutant mice were crossed by in vitro fertilization. In methylcellulose clonal culture of fetal liver (FL) cells of generated hGMR-expressing EPOR-/- embryos at embryonic day (E) 12.5 of gestation, hGM-CSF stimulated erythroid colony formation under serum-containing and serum-free conditions. Analysis of globin expression in individual erythrocyte-containing colonies formed from E12.5 FL cells showed that hGM-CSF supports primitive and definitive erythropoiesis even in EPOR-/- embryos. In comparison of activities between hGM-CSF and EPO in hGMR-expressing EPOR+/+ embryos, the 2 substances supported the formation of similar numbers of erythroid colonies in clonal culture of E12.5 FL cells; enhanced adult, but not embryonic, globin synthesis; and induced increase of GATA-1 expression and decrease of erythroid Kruppel-like factor and cMyb expression in the FL cells. On the other hand, in E8.0 yolk sac erythropoiesis, both substances had a similar effect on erythroid colony formation, but hGM-CSF induced an increase of β-major globin expression, while EPO did not. All together, the results of the present study demonstrated that hGM-CSF can stimulate the proliferation and differentiation of primitive and definitive erythroid cells independently of EPOR signal if they express hGMR, and the activity is comparable to that of EPO in definitive, but not primitive, erythropoiesis.