Even though eukaryote health relies on beneficial symbionts, host defenses targeting pathogens create substantial obstacles for the establishment of these essential partnerships. To reveal mechanisms of symbiotic adaptation, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri through Euprymna scolopes squid light organs. Serial passaging of V. fischeri populations through squid hosts produced eight distinct mutations in the binK sensor kinase gene that conferred an exceptional selective advantage demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion behavior which was mediated by symbiotic polysaccharide (Syp). binK variation also produced metabolic convergence with native symbionts, and altered quorum sensing and luminescence. Preexisting coordination of symbiosis traits facilitated an efficient solution where altered function of a regulator was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations.