To examine the influence of the hematopoietic microenvironment on hematopoietic cell proliferation and differentiation during the yolk sac phase of hematopoiesis, we have recently established cell lines from embryonic yolk sac visceral endoderm (YSE) and mesoderm (YSM). In the present experiments, we compared in vitro growth of adult murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in coculture with YSE- and YSM-derived or adult bone marrow stromal cell lines. Whereas both yolk sac-derived and adult stromal cell lines supported the proliferation of HPP-CFC during coculture, YSE- and YSM-derived cells stimulated a significant increase in total HPP-CFC compared with adult bone marrow stromal cell lines. Conditioned media from both YSE- and YSM-derived cell lines also stimulated the growth of HPP-CFC in vitro, but only in combination with exogenous recombinant hematopoietic growth factors. Although multiple hematopoietic growth factor mRNAs were detected in the yolk sac-derived cells by polymerase chain reaction, only macrophage colony-stimulating factor (M-CSF) activity was detected in conditioned media using an enzyme-linked immunosorbent assay. A neutralizing polyclonal antibody against M-CSF did not diminish the YSE- or YSM-derived cell line conditioned media promotion of HPP-CFC colony formation. These results suggest that murine yolk sac-derived cell lines produce a novel soluble factor(s) that recruits primitive bone marrow hematopoietic cells to grow in vitro in response to a combination of hematopoietic growth factors.