The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional (3-D) microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) (PDMS) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 µm in width at a constant depth of 1 µm. During 4 h time-lapse observation in the absence of flow, the majority of the pre-aligned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm²), irrespective of groove direction with respect to flow. After 4 h exposure to high shear stress (58 dyn/cm²) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the cells’ migration direction under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.