Endothelial cell (EC) migration plays an important role in embryonic vasculogenesis, angiogenesis, and wound healing. EC migration can be regulated by extracellular matrix (ECM) and hemodynamic forces through haptotaxis (induced by an ECM gradient) and mechanotaxis (induced by mechanical forces). Previously, the effects of haptotaxis or mechanotaxis alone on EC migration have been studied; however, the dual effect of haptotaxis and mechanotaxis on EC migration is not known. We developed a micropatterning technique to generate step changes of collagen surface density to monitor haptotactic EC migration. To investigate the crosstalk between haptotaxis and mechanotaxis on EC migration, we used an in vitro flow system to apply a well-defined fluid shear stress on ECs cultured on the micropatterned collagen. The study on the effects of haptotaxis and mechanotaxis on EC migration will provide a rational basis for promoting vascular wound healing, angiogenesis, and vascularization in engineered tissues.