Environmental factors are instrumental in maintaining a healthy vasculature. Oxygen tension is higher in arteries than in veins and thus has the potential to be an instructive signal in arterial/venous specification. EphrinB2 is specifically expressed in arteries and required during embryonic vessel formation. In this study, we show that expression of ephrinB2 is oxygen dependent. Mutagenesis of hypoxia-responsive elements and transactivation experiments determined this regulation to be achieved in a hypoxia-inducible factor independent manner. MAZ and Sp1 are known to regulate transcription together and have been shown to bind to the same sites within promoters. Chromatin immunoprecipitation confirmed that binding of Sp1 to the ephrinB2 promoter was favored compared to MAZ under hypoxic relative to normoxic conditions. Furthermore, siRNA mediated knockdown of Sp1 attenuated this hypoxic response. These results indicate that hypoxia drives arterial differentiation by increasing ephrinB2 expression in endothelial cells through Sp1 activation.