Blood-borne bacteria like the Lyme disease pathogen Borrelia burgdorferi cause infection by migrating across the vascular endothelial barrier into target tissues. The mechanisms by which this occurs are poorly understood, largely because model systems inadequately mimic the in vivo environment or are too inefficient to dissect mechanisms. This unmet need is addressed in this thesis by the development of a microfluidic system and live cell imaging methods to model and study transendothelial migration of bacteria in vitro under physiologically relevant conditions. Real-time transmigration kinetics of B. burgdorferi across intact endothelium were obtained, for the first time, under static and flow conditions. Validation studies confirmed that B. burgdorferi transmigrate actively, with similar kinetics to conventional Transwell systems under static conditions. Additionally, physiological shear stress conditions appeared not to significantly alter transmigration kinetics. These data were uniquely obtainable with the microfluidic platform, supporting its utility for studying extravasation of blood-borne pathogens of worldwide significance.