Flow-induced parallel alignment of endothelial cells (ECs) is replicated in vitro in macroscale parallel plate flow chambers (PPFCs), but with increasing application of microfluidic platforms to study EC biology and model ECs in organ-on-a-chip platforms, there is a need to better understand flow regulation of ECs in microscale platforms. Previous work in our labs found that EC monolayers confined in microchannels and PDMS stencils aligned perpendicular to the direction of flow in contrast to parallel alignment in macroscale PPFCs. The goal of this thesis was to elucidate the microfluidic culture parameters that condition for the confined EC phenotype. Perpendicular alignment to flow was observed in human umbilical vein ECs (HUVECs) across all tested physical and geometrical conditions and suggested that spatial control provides cues for a confined EC phenotype. This work demonstrated that perpendicular alignment to flow is a robust response in microscale-cultured ECs.