The binary association between the squid, Euprymna scolopes, and its symbiont, Vibrio fischeri, serves as a model system to study interactions between beneficial bacteria and the innate immune system. Previous research demonstrated that binding of the squid's immune cells, hemocytes, to V. fischeri is altered if the symbiont is removed from the light organ, suggesting that host colonization alters hemocyte recognition of V. fischeri. To investigate the influence of symbiosis on immune maturation during development, we characterized hemocyte binding and phagocytosis of V. fischeri and nonsymbiotic Vibrio harveyi from symbiotic (sym) and aposymbiotic (apo) juveniles, and wild-caught and laboratory-raised sym and apo adults. Our results demonstrate that while light organ colonization by V. fischeri did not alter juvenile hemocyte response, these cells bound a similar number of V. fischeri and V. harveyi yet phagocytosed only V. harveyi. Our results also indicate that long-term colonization altered the adult hemocyte response to V. fischeri but not V. harveyi. All hemocytes from adult squid, regardless of apo or sym state, both bound and phagocytosed a similar number of V. harveyi while hemocytes from both wild-caught and sym-raised adults bound significantly fewer V. fischeri, although more V. fischeri were phagocytosed by hemocytes from wild-caught animals. In contrast, hemocytes from apo-raised squid bound similar numbers of both V. fischeri and V. harveyi, although more V. harveyi cells were engulfed, suggesting that blood cells from apo-raised adults behaved similarly to juvenile hosts. Taken together, these data suggest that persistent colonization by the light organ symbiont is required for hemocytes to differentially bind and phagocytose V. fischeri. The cellular immune system of E. scolopes likely possesses multiple mechanisms at different developmental stages to promote a specific and life-long interaction with the symbiont.