P. aeruginosa is an opportunistic pathogen that occupies diverse environmental niches and is capable of causing a range of infections in humans. This versatility suggests that it has sophisticated mechanisms to sense and respond to the surrounding microenvironment. Two-component sensors are commonly used by bacteria to sense and respond to environmental stimuli and Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria. We took advantage of a non-redundant transposon library and a recently characterized vertebrate model host, Danio rerio, that is amenable to higher throughput analysis than mammalian models, to systematically test the role of 60 two-component sensors that are required for P. aeruginosa virulence in acute infection. We found that the sensor kinase KinB is required for acute infection in zebrafish embryos and regulates a number of virulence related phenotypes in a manner independent of its kinase activity and its known response regulator, AlgB. Thus, the regulation of virulence by KinB highlights the increasing recognition of non-canonical two-component signaling mechanisms.