The major arteries and veins of the vertebrate circulatory system are formed early in embryonic development, before the onset of circulation, following de novo aggregation of “angioblast” progenitors in a process called vasculogenesis. Initial embryonic determination of artery or vein identity is regulated by variety of genetic factors that work in concert to specify endothelial cell fate, giving rise to 2 distinct components of the circulatory loop possessing unique structural characteristics. Work in multiple in vivo animal model systems has led to a detailed examination of the interacting partners that determine arterial and venous specification. We discuss the hierarchical arrangement of many signaling molecules, including Hedgehog (Hh), vascular endothelial growth factor (VEGF), Notch, and chicken ovalbumin upstream-transcription factor II (COUP-TFII) that promote or inhibit divergent pathways of endothelial cell fate. Elucidation of the functional role of these genetic determinants of blood vessel specification together with the epigenetic factors involved in subsequent modification of arterial–venous identity will allow for potential new therapeutic targets for vascular disorders.