Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, we performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, we classified 646 identified genes into phenotypic and functional groups and unveiled a comprehensive set of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, our data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlight the intricate regulation of protein synthesis during differentiation. Display omitted •An in vivo RNAi screen identifies 646 genes involved in germline stem cell biology•Ribosome assembly and eIF4E modulate the final step of stem cell cytokinesis•Stem cell differentiation relies on pathways promoting global protein synthesis•rRNA and protein synthesis are uncoupled during stem cell differentiation The complex gene regulatory networks that control stem cell behavior are only partially understood. Sanchez et al. describe an unbiased in vivo RNAi screen and provide a formative dissection of the gene pathways required for germline stem cell development.