Tumor-associated myeloid cells are believed to promote tumor development by stimulating tumor growth, angiogenesis, invasion, and metastasis. Tumor-associated myeloid cells that coexpress endothelial and myeloid markers represent a proangiogenic subpopulation known as vascular leukocytes. Recently, we and others had shown that tumor-derived TNFα promotes local tumor growth and vascularity. Our data suggested that tumor growth is in part due to TNFα-mediated increased numbers of tumor-associated vascular leukocytes (i.e., myeloid–endothelial biphenotypic cells). The work detailed herein explored the mechanism by which TNFα mediates endothelial differentiation of myeloid cells. Our studies showed that fibronectin is a robust facilitator of endothelial differentiation of blood mononuclear cells in vitro. We have found that TNFα treatment of monocytes significantly increased expression of α₅β₁ integrin, a major fibronectin receptor enriched on endothelial cells, leading to a consequent fourfold increase in fibronectin adhesion. Furthermore, TNFα-treated monocytes upregulated expression of endothelial markers, flk-1(VEGFR2/KDR) and VE-cadherin. Integrin α₅ subunit inhibitory antibodies blocked adhesion to fibronectin as well as consequent upregulation of flk-1 and VE-cadherin transcripts, implying a role for outside-in signaling by the α₅β₁ integrin after binding fibronectin. Finally, treatment of mouse tumors with anti-α₅ antibodies reduced accumulation of tumor vascular leukocytes in vivo. Our studies suggest that tumor cell–derived TNFα constitutes a tumor microenvironment signal that promotes differentiation of tumor-associated monocytes toward a proangiogenic/provasculogenic myeloid–endothelial phenotype via upregulation of the fibronectin receptor α₅β₁. Mol Cancer Res; 9(6); 702–11. ©2011 AACR.