Anamorsin (AM), an anti-apoptotic molecule, confers factor-independent survival on hematopoietic cells. AM-deficient (AM–/–) mice are embryonic lethal due to a defect in definitive hematopoiesis. However, the significance of AM in embryonic hematopoiesis remains unknown. This study characterized the hematopoietic defects in AM–/– fetal livers. The AM–/– fetal liver displayed significantly reduced numbers of c-Kit+ Sca-1+ Lin– (KSL) cells. An in vitro colony-forming unit assay showed that fetal liver cells isolated from AM–/– embryos gave rise to fewer colonies in all cell types. Primary and secondary transplantations with AM–/–cells were markedly inefficient in the reconstitution of all lineages. Furthermore, the limiting dilution assay revealed that fetal liver hematopoietic stem cell function was impaired due to AM deficiency. The reconstitution activity in AM–/– hematopoietic stem cells (HSCs) was markedly reduced in all lineages. Furthermore the limiting dilution assay revealed that the number of fetal liver HSCs was reduced due to AM deficiency. Retrovirus-mediated AM expression rescued the defective hematopoietic colony-forming activities of AM–/– KSL cells. We also investigated the effects of AM deficiency on fetal liver stromal cells, which support hematopoiesis. Interestingly, primary stromal cell cultures from wild-type fetal liver supported the growth of AM–/– KSL cells, but stromal cultures from AM–/– fetal liver provided little support of wild-type KSL cell growth. These results demonstrated that AM was essential for both autonomous and extrinsic regulation of fetal liver hematopoiesis. This study provided new insight into the molecular regulation of hematopoiesis.