Many stem cell types have been shown to differentiate into endothelial cells (ECs), however, their specification to arterial or venous endothelium remains unexplored. We tested whether a specific arterial or venous EC fate could be induced in human Multipotent Adult Progenitor Cells (hMAPCs) and AC133(+) cells (hAC133(+)). In vitro, in the presence of VEGF165, hAC133(+) cells only adopted a venous and microvascular EC phenotype, while hMAPCs differentiated into both arterial and venous ECs, possibly because hMAPCs expressed significantly more sonic hedgehog (Shh) and its receptors, as well as Notch 1 and 3 receptors and some of their ligands. Accordingly, blocking either of those pathways attenuated in vitro arterial EC differentiation from hMAPCs. Complementarily, stimulating these pathways by addition of Delta-like 4 (Dll-4), a Notch ligand, and Shh to VEGF165 further boosted arterial differentiation in hMAPCs both in vitro and in an in vivo matrigel model. These results represent the first demonstration of adult stem cells with the potential to be differentiated into different types of ECs in vitro and in vivo and provide a useful human model to study arterio-venous specification.