Symbiotic associations often enhance hosts' physiological capabilities, allowing them to expand into restricted terrains, thus leading to biological diversification. Stable maintenance of partners is essential for the overall biological system to succeed. The viviparous tsetse fly (Diptera: Glossinidae) offers an exceptional system to examine factors that influence the maintenance of multiple symbiotic organisms within a single eukaryotic host. This insect harbours three different symbionts representing diverse associations, coevolutionary histories and transmission modes. The enterics, obligate mutualist Wigglesworthia and beneficial Sodalis, are maternally transmitted to the intrauterine larvae, while parasitic Wolbachia infects the developing oocyte. In this study, the population dynamics of these three symbionts were examined through host development and during potentially disruptive events, including host immune challenge, the presence of third parties (such as African trypanosomes) and environmental perturbations (such as fluctuating humidity levels). While mutualistic partners exhibited well-regulated density profiles over different host developmental stages, parasitic Wolbachia infections varied in individual hosts. Host immune status and the presence of trypanosome infections did not impact the steady-state density levels observed for mutualistic microbes in either sex, while these factors resulted in an increase in Wolbachia density in males. Interestingly, perturbation of the maternal environment resulted in the deposition of progeny harbouring greater overall symbiont loads. The regulation of symbiont density, arising from coadaptive processes, may be an important mechanism driving inter-specific relations to ensure their competitive survival and to promote specialization of beneficial associations.