Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissues. In the process EPCs are exposed to the shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress promotes differentiation of EPCs into mature endothelial cells. In this study we investigated whether EPCs differentiate into arterial or venous endothelial cells in response to shear stress. When cultured EPCs derived from human peripheral blood were exposed to controlled levels of shear stress in a flow-loading device, the mRNA levels of the arterial endothelial cell markers ephrinB2, Notch1/3, Hey1/2, and ALK1 increased, but the mRNA levels of the venous endothelial cell markers EphB4 and NRP2 decreased. Both the ephrinB2 increase and the EphB4 decrease were shear-stress- rather than shear-rate-dependent. EphrinB2 protein was increased in shear-stressed EPCs, and the increase in ephrinB2 expression was due to activated transcription and not mRNA stabilization. Deletion analysis of the ephrinB2 promoter indicated that the cis-element (shear stress response element) is present within 106 bp 5' upstream from the transcription initiation site. This region contains the Sp1 consensus sequence, and a mutation in its sequence decreased the basal level of transcription and abolished shear stress-induced ephrinB2 transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that shear stress markedly increased binding of Sp1 to its consensus sequence. These results indicate that shear stress induces differentiation of EPCs into arterial endothelial cells by increasing ephrinB2 expression in EPCs through Sp1 activation.