Objective: Two competing hypotheses can be formulated regarding the origin of canine hemangiosarcoma (HSA). One states HSA originates from differentiated vascular endothelial cells that undergo mutations which endow them with malignant potential. The other states HSA originates from transformed hemangioblastic stem cells. This study was designed to begin to distinguish between these possibilities, as well as to test if flow cytometry was sufficiently sensitive to detect malignant cells in blood samples from dogs with HSA. Methods: We used multiparameter flow cytometry to examine expression of cell-surface determinants associated with hematopoietic precursors (c-kit, CD34, CD133, CD45) or with lineage-committed cells (CD3, CD11b, CD14, CD21, CD105, CD146, αvβ3-integrin) in HSA cell lines and in blood samples from healthy dogs or dogs with HSA. Results: The data show that HSA cells coexpress surface markers associated with hematopoietic precursors and with commitment to endothelial lineage, providing a means to identify their presence in circulation and distinguish them from normal or malignant white blood cells. The percentage of cells that coexpressed these markers ranged from 0.5 to 1.25% for HSA dogs, and was less than 0.3% for unaffected dogs or dogs with HSA that had the tumors removed within 48 hours prior to obtaining samples. Conclusions: The results place the ontogeny of HSA with multipotential bone marrow-derived stem cells whose progeny arrest differentiation at the hemangioblast or angioblast stage. In addition, these expression patterns may assist to confirm an HSA diagnosis, monitor minimal residual disease, and detect the disease in early stages. © 2006 International Society for Experimental Hematology.