Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance.