The development processes of arteries and veins are fundamentally different, leading to distinct differences in anatomy, structure, and function as well as molecular profiles. Understanding the complex interaction between genetic and epigenetic pathways, as well as extracellular and biomechanical signals that orchestrate arterial venous differentiation, is not only critical for the understanding of vascular diseases of arteries and veins but also valuable for vascular tissue engineering strategies. Recent research has suggested that certain transcriptional factors not only control arterial venous differentiation during development but also play a critical role in adult vessel function and disease processes. This review summarizes the signaling pathways and critical transcription factors that are important for arterial versus venous specification. We focus on those signals that have a direct relation to the structure and function of arteries and veins, and have implications for vascular disease processes and tissue engineering applications.