Endothelial cells (ECs) line all blood contacting surfaces within the body and are exposed to hemodynamic shear stresses in vivo. To investigate EC biology in vitro, researchers have primarily used either conventional static systems such as well plates, or flow systems, such as parallel plate flow chambers. These macroscale platforms, although convenient, use large quantities of reagents and/or are not physiologically relevant. Recently, microfluidics has emerged as an alternative approach to study vascular biology. To demonstrate the usefulness of microfluidics in studying EC biology, four microfabricated platforms were developed and characterized. We demonstrated for the first time that primary cells, specifically multiple EC types, could be cultured and analyzed on a digital microfluidic platform. ECs patterned on a device using a fluorocarbon liftoff technique displayed both appropriate morphologies and functions. Second, a membrane microfluidic device was developed to mimic key components of the vascular microenvironment. Complex interactions between shear stress and tumour necrosis factor-α stimulation on monocyte adhesion to ECs and preferential transmigration of monocytes in response to a chemoattractant were investigated. This platform was subsequently advanced to incorporate a mural cell-laden three-dimensional gelatin methacrylate hydrogel beneath a confluent EC monolayer to more fully mimic the vascular/valvular environment. The presence of ECs suppressed valvular interstitial cell pathological differentiation to α-smooth muscle actin-expressing myofibroblasts, an effect that was enhanced when the endothelium was exposed to flow-induced shear stress. Lastly, EC phenotypes under static and flow conditions were analyzed in simple, straight, rectangular microfluidic channels to better characterize EC behaviour in microchannels. Interestingly, ECs were observed to align perpendicular to the direction of flow in microchannels of widths between 300 – 20000 µm. Vascular ECs align parallel to flow in vivo, and have never been reported to align perpendicular to flow in vitro. A comparison of EC phenotype between microchannel and macroscale culture showed differences in the arrangement of cell-cell junctions suggesting a possible role for junctional proteins in mediating perpendicular versus parallel alignment to flow. Overall, the work presented in this thesis demonstrates the utility of microfluidic platforms for EC biology and opens a discussion on potential new mechanisms of endothelial mechanotransduction.