Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis and interaction with myocardium in the heart. We also found that MEKK3 is required for angiogpoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild type and Mekk3-deficient tumor-like embryonic stem cell lines, and transplanted them subcutaneously into nude mice to assess their ability to grow and to induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as the control Mekk3 wild type tumors, and were also capable of inducing tumor angiogenesis. In addition, we found no difference in the production of VEGF in Mekk3-deficient either tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation, survival, and is required for endothelial cells to interact with myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis.