The vertebrate vascular system is essential for the delivery and exchange of gases, hormones, metabolic wastes and immunity factors. These essential functions are carried out in large part by two types of anatomically distinct blood vessels, namely arteries and veins. Previously, circulatory dynamics were thought to play a major role in establishing this dichotomy, but recently it has become clear that arterial and venous endothelial cells are molecularly distinct even before the output of the first embryonic heartbeat, thus revealing the existence of genetic programs coordinating arterial-venous differentiation. Here we review some of the molecular mechanisms involved in this process.