An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1 alpha, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the sternness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors. (Am J Pathol 2012,180:1294-1307; DOI: 10.1016/j.ajpath.2011.11.035)