Understanding how organisms adapt to changes in their environment at both the genotypic and phenotypic level is a fundamental challenge of evolutionary biology. In Drosophila melanogaster a key germline stem cell differentiation factor which is required for gametogenesis, bag of marbles (bam) shows signatures of adaptive evolution between its sibling species D. simulans, but not in several outgroup species. However, the functional consequences and drivers of the genetic signals of adaptation at bam remain unclear. Bam is a novel protein to a dipteran lineage containing Drosophila, therefore it is possible bam has functionally diversified across the Drosophila genus, only gaining its known function in the lineage leading to D. melanogaster and D. simulans, thereby resulting in adaptive evolution for this function. Additionally, it is possible that bam quickly evolved a role in GSC differentiation in Drosophila, but that function has been shaped by germline conflicts in specific lineages. Notably, the endosymbiotic bacteria Wolbachia resides in the germline and rescues the fertility defect of a bam partial loss-of-function mutant in D. melanogaster. In this dissertation I asked if bam function is conserved across five representative Drosophila species to test if a novel role in germline stem cell differentiation could be driving the adaptive evolution of bam. I also asked if Wolbachia variation impacts the rescue of the bam partial loss-of- function phenotype to further understand the dynamics of the bam and Wolbachia interaction in D. melanogaster. I found that bam function is not fully conserved across these species, and its function may evolve on a relatively short time scale. I also found an effect of Wolbachia variation on the degree of the bam fertility rescue. My findings indicate that the episodic signals of adaptive evolution at bam are unlikely to be driven by a single gain in function for bam as a GSC differentiation factor, and that we can use variation in Wolbachia to further define the biological mechanism of its interaction with bam to understand how Wolbachia impacts bam evolution.