Germline stem cell (GSC) regulating genes are developmentally critical in Drosophila and have been assumed to be highly conserved functionally. However, recent comparative functional and evolutionary results suggest that bag of marbles (bam) and perhaps other GSC genes may have an unexpected rate of turnover in their functions and even presence in different Drosophila species. Such a pattern of evolution suggests a surprising evolutionary flexibility in the genes that regulate GSC maintenance and differentiation. Recent results in our lab demonstrate that bam’s essential role as the master switch of GSC differentiation described for D. melanogaster is not required in either females or males in D. teissieri and in males of D. ananassae. This begs the question of whether bam’s essential role is an evolutionarily recent acquisition in D. melanogaster and its close relatives. To test this, I am generating bam nulls in two species, D. pseudoobscura and D. americana. Results from these nulls will more precisely define the direction of the acquisition of bam’s essential GSC function in D. melanogaster. I am also interrogating GSC gene functional flexibility more broadly by generating a GSC gene ortholog dataset for a representative subset of Drosophila and relevant outgroup species. Resources for assessing gene orthology across species incorporate sequencing data of highly variable quality, necessitating direct assessment of predicted ortholog absences. This direct assessment will be executed using species and gene specific PCR validation. Identifying and verifying GSC gene ortholog presence or absence will reveal key functional differences in a developmentally critical regulatory network between closely related species which would form the basis of targeted functional analyses. The dataset will also provide insight into whether particular subsets of genes based on function, structure, or interactions, tend to be absent or conserved.