The remarkable capacity of stem cells to balance self-renewal and differentiation is tightly coordinated by the niche in which they reside. The niche comprises a complex environment of cellular and non-cellular components, providing structural and signalling inputs to control stem cell fate in a spatially precise manner. The Drosophila ovarian germline stem cell (GSC) niche provides an excellent model to examine niche communication; with 2-3 germline stem cells surrounded by spatially distinct populations of somatic support cells (cap cells, terminal filament cells and escort cells). Depletion of the transcriptional repressor Half-pint (Hfp) specifically in the escort cells comprising the niche, drives niche cell escape from the stem cell compartment. Moreover, the mislocalised escort cells generate ectopic niches capable of supporting germline stem cell renewal and differentiation. This raises the question: what are the endogenous mechanisms for maintaining escort cell positioning? Intriguingly, our escort cell/niche-specific genome-wide binding data (Targeted-DamID) demonstrated direct Hfp binding to gene targets encoding extracellular matrix-receptor interactions. Using high-resolution single cell sequencing we are now investigating how Hfp-knockdown alters gene expression in the niche, and the neighbouring germline lineage, prior to escort cell escape. We further aim to determine how ectopic niches, driven by Hfp-knockdown, maintain germline stem cell renewal and differentiation. To answer this question, we are using advanced spatial multi-omics technology to determine the transcriptome of newly formed Hfp-KD ectopic stem cell niches. Specifically, we are applying spatial enhanced resolution omics-sequencing (Stereo-seq) to map cell identities and gene expression changes across endogenous and ectopic ovarian stem cell niches. Thus, we will identify mechanisms of escort cell escape and determine the minimal signalling requirements for stem cell fate control. Understanding how stem cells escape normal regulation will shed light on diseases of abnormal stem cell behaviour, such as cancer.