Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center towards the leading edge of migrating cells in a Cdc42 dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to four days, but not shear stress-induced morphology. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and induced an increase in the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant negative Par6, a major downstream effector of Cdc42-induced polarity. Shear stress induced directed migratory polarity is modulated by exogenous growth factors and dependent on Par6 activity and shear stress direction.