There is intense interest in the circuits that guide stem cell behavior. In many of our tissues, these stem cell regulatory circuits are controlled by the niches that house the stem cells. it is not understood how the niche is specified and assembled in a tissue, and then how it executes control over the stem cell pool. Understanding these interactions will be crucial to the use of stem cells in regenerative medicine. This proposal utilizes one of the most well-understood stem cell-niche systems, the Drosophila testis. Here, a small group of cells (hub cells) act as part of the niche, leading to the activation of signaling pathways in adjacent cells. In this way, nearby somatic cells take on cyst stem cell fate (CySC), while nearby germline cells, intermingled with these CySCs, take on germline stem cell fate (GSC). Importantly, these two distinct stem cell types must coordinate their production of daughter cells for spermatogenesis to occur properly. Over the last funding cycle, we made fundamental breakthroughs in live- imaging both niche assembly and niche function. Here, we capitalize on this unprecedented level of resolution to tackle the mechanisms that underlie niche assembly and function. Hub formation, and the attendant attachment of stem cells, is the major architectural event of gonadogenesis. The specification and placement of hub cells among somatic gonadal precursors (SGPs) generates an anteriorly-anchored proliferation center that will drive spermatogenesis in a polarized manner. To generate that polarity, a subset of pre-hub cells must respond to positioning cues. However, neither those cues nor their source are currently known. This proposal seeks to define those cues and how they work. In many of our tissues, niches house multiple stem cell types, just as does the Drosophila testis niche. Thus, to maintain tissues the behavior of the multiple resident stem cell types must be coordinated. However, in no case do we understand how. This proposal will address this essential question.