Abstract: The earliest hematopoiesis originates in the yolk sac, which forms prior to blood circulation and develops differently in primates and mice. Despite the increasing appreciation of the yolk sac hematopoiesis for its life-long contribution to the adult hematopoietic system and regulatory roles in organogenesis, insights into cross-species differences in it, especially before the onset of blood circulation, are limited. Here, we constructed an integrative cross-species transcriptome atlas of the pre-circulation hematopoiesis between human, monkey (Macaca fascicularis), and mouse. The cross-species analysis identified similar populations between primates and mice. However, more differentiated lineages of myeloid, erythroid, and megakaryocytic populations were observed in pre-circulation primates compared to mice. Specifically, macrophages with expression of SPP1 are already detectable in primates before the onset of blood circulation, but are absent in mice. Cell-cell communication analysis revealed that CSF1+ extraembryonic mesoderm might contribute to supportive niche for macrophage generation and ligand-receptor interactions from macrophages to other cell populations in human yolk sac. Interestingly, the pre-circulation SPP1+ macrophages showed similar hallmark signatures to the specific subset of macrophages which is capable of positively regulating hematopoietic stem cell generation. Our work has provided cross-species resource and valuable insights into human pre-circulation yolk sac hematopoiesis and the theoretical basis for the regeneration of functional blood cells.