The stable inheritance of gene expression programs during development is required to generate and maintain cellular diversity from a single genome. The Polycomb Repressive Complex 2 (PRC2) is an essential chromatin modifier that maintains gene repression by catalyzing the trimethylation of lysine 27 on histone (H3K27me3), which leads to the formation of facultative heterochromatin. During development, PRC2 is capable of establishing new cell type-specific domains of H3K27me3 in addition to maintaining existing ones, but how PRC2 accomplishes this dual function is unclear. Two different versions of PRC2 exist, termed PRC2.1 and PRC2.2, based on the mutually exclusive association of the PRC2 catalytic core with different cohorts of accessory factors. The potentially unique roles of PRC2.1 and PRC2.2 in shaping PRC2 activity are not well understood, due to contrasting reports in the literature describing both their functional redundancy and differential requirements in different cell types. The overarching goal of my thesis was to interrogate the mechanisms regulating the establishment of repressive H3K27me3 domains by PRC2. In chapter 2, I dissected the roles of the two PRC2 holocomplexes—PRC2.1 and PRC2.2—in this process by employing auxin-inducible protein degradation of PRC2.1 and PRC2.2 subunits during the differentiation of mouse embryonic stem cells to neural progenitors. This revealed that PRC2.1 and PRC2.2 regulate largely non-overlapping gene sets with distinct chromatin and expression dynamics, suggesting that PRC2.1 and PRC2.2 play distinct roles in mediating the maintenance and establishment of Polycomb repression during differentiation. In chapter 3, I carried out a genome-wide CRISPR knockout screen to identify novel regulators of H3K27me3 establishment, which recovered candidate regulators with links to RNA processing, nuclear architecture, DNA repair, and transcription. Overall, my results are the first to propose a division of labor between the two PRC2 assemblies in regulating H3K27me3 maintenance and establishment, and reveal intriguing potential connections between PRC2 and other chromatin machineries in the establishment of H3K27me3-marked facultative heterochromatin.