ABSTRACT INTESTINAL DIFFERENTIATION: FROM CHROMATIN TO THE NICHEHannah M. Kolev Klaus H. Kaestner To continuously replenish the intestinal epithelium (IE), multipotent intestinal stem cells (ISCs) balance self-renewal with differentiation towards mature cell lineages. Investigating the extrinsic and intrinsic mechanisms guiding ISC differentiation is a fundamental component of intestinal biology and critical for advancing our understanding of gastrointestinal health. In this dissertation, I present two studies addressing limitations in our understanding of ISC differentiation, the first through the lens of the extrinsic ISC niche and the second through the lens of the intrinsic epigenetic mechanisms guiding cell fate decisions. Current investigations of Foxl1+ telocytes, a mesenchymal cell population that functions as the ISC niche, are limited by the lack of tractable mouse models enabling the isolation and manipulation of this cell population. In the first study, I address this limitation by characterizing a novel Foxl1-CreERT2-2A-tdTomato (Foxl1CreER-tdTom) mouse model in which the endogenous Foxl1 promoter drives expression of inducible Cre recombinase and a fluorescent reporter. I perform validation experiments demonstrating that Foxl1CreER-tdTom mice drive efficient Cre activity in Foxl1+ telocytes along the gastrointestinal tract. Furthermore, I derive a novel floxed Glp2r allele, that when crossed to Foxl1CreER-tdTom mice, will elucidate whether Foxl1+ telocytes mediate GLP-2 signaling in the intestine. Together, these mouse models represent valuable tools for enhancing our understanding of Foxl1+ telocytes and the ISC niche. Despite significant progress in defining the epigenomic landscape of the IE, our understanding of the precise epigenetic mechanisms regulating ISC differentiation remains limited. In the second study, I address this knowledge gap by investigating the function of KDM6A and KDM6B (KDM6A/B), two H3K27me3 demethylases, in the IE. By removing Kdm6a/b expression from the IE, I demonstrate that KDM6A/B are dispensable for cell lineage allocation during intestinal homeostasis and development. Through transcriptomic and epigenomic approaches, I find that KDM6A/B maintain optimal gene expression and H3K27me3 levels at a subset of genes in the IE. Furthermore, I identify a role for KDM6A/B in regulating expression of the Paneth cell gene signature as well as expression and localization of a key Paneth cell enzyme. Together, this study demonstrates that KDM6A/B support the full transcriptomic and epigenomic landscape of the IE.