Abstract Although shear flow in the human body restricts colonization by bacterial pathogens, our understanding of how bacterial pathogens sense and respond to shear flow is limited. Recently, I combined microfluidics and transcriptomics to discover that the human pathogen, Pseudomonas aeruginosa, senses shear flow through a novel process, which I named rheosensing. Rheosensing uses the sigma- factor FroR and anti-sigma factor FroI to tune gene expression to the speed of shear flow. Here, I propose a systems-level approach to characterize the regulatory mechanisms of rheosensing. First, I will use microfluidic-based transcriptomics to measure genome-wide changes in gene expression while independently modifying the three parameters of shear flow: flow rate, channel dimensions, and viscosity. Second, I will determine the regulatory targets of the rheosensing regulators FroR and FroI. Third, I will couple fluorescence-activated cell sorting (FACS) with transposon sequencing (Tn-seq) to identify additional sensory and regulatory proteins that control rheosensing. Together, these aims will provide genome-wide characterization of the targets, signaling pathways, and flow sensors that control rheosensing. As rheosensing is likely important for host colonization, this study will provide new insight into the virulence of P. aeruginosa in host-relevant shear flow. Together, the research and career development plans I outline here will help me obtain a faculty position at a research university, launch my independent research program, and allow for a seamless transition to the next stage of my career.