The role of vasculogenesis as opposed to angiogenesis in tumor formation has been little explored genetically. Endothelial cells that lack the MEK kinase MEKK3 cannot form vessels. In this study, we employed mice with hematopoietic deletions of the Mekk3 gene to evaluate the importance of vasculogenesis in the formation of Ewing's sarcoma tumors. Bone marrow cells (BM) from LacZ(+) Mekk3-deficient conditional knockout mice (Mekk3(Deltaflox/-) mice) were transplanted into irradiated nude mice before injection of Ewing's sarcoma cells. Because the grafted Mekk3(Deltaflox/-) BM cells cannot contribute to vessel development in the same way as the host Mekk3(+/+) endothelial cells, angiogenesis is normal in the model whereas vasculogenesis is impaired. Four weeks after BM transplant, Ewing's sarcoma TC71 or A4573 cells were injected, and tumor growth and vessel density were compared. Strikingly, chimeric mice transplanted with Mekk3(Deltaflox/-) BM exhibited a reduction in tumor growth and vessel density compared with mice transplanted with Mekk3(Deltaflox/+) BM cells. Mekk3(Deltaflox/-) cells that were LacZ positive were visualized within the tumor; however, few of the LacZ(+) cells colocalized with either CD31(+) endothelial cells or desmin(+) pericytes. Quantification of double-positive LacZ(+) and CD31(+) endothelial cells or LacZ(+) and desmin(+) pericytes confirmed that chimeric mice transplanted with Mekk3(Deltaflox/-) BM were impaired for tumor vessel formation. In contrast, siRNA-mediated knockdown of Mekk3 in TC71 Ewing's sarcoma cells had no effect on tumor growth or vessel density. Our findings indicate that vasculogenesis is critical in the expansion of the tumor vascular network.