In mammalian development, the process of gametogenesis begins with a group of germline precursors known as primordial germ cells (PGCs), which emerge shortly after embryo implantation. These PGCs undertake a migration through the developing hindgut to ultimately reach and populate the embryonic gonads. In vitro modeling of this complex developmental process offers a non-invasive method to study the molecular mechanisms governing gametogenesis. This is particularly significant for understanding the genetic and epigenetic underpinnings of infertility, which can inform advancements in assisted reproductive technologies (ART), including gene correction techniques and the optimization of in vitro gametogenesis (IVG). My thesis will concentrate on refining this in vitro model for generating mouse PGCs, aiming to make the process more efficient and scalable. An important aspect of my research will involve utilizing CRISPR technology for perturbation screens, designed to identify key epigenetic regulators that play a crucial role in PGC development. By enhancing our understanding of these regulators and the overall process of PGC development, my research could provide valuable insights and tools for improving ART and IVG techniques, ultimately aiding in the treatment of infertility and related reproductive issues. In Chapter 1 of my thesis, I provide a comprehensive overview of the current understanding in several key areas related to mammalian PGC development. This includes an in-depth examination of IVG, the epigenetic mechanisms at play during PGC development, and the various applications of the PGC-like cell (PGCLC) platform. The primary objective of this chapter is to collect and present all relevant information concerning the early stages of embryonic germline development, with a particular focus on the period preceding sex determination. By compiling this information, the chapter aims to offer a clear picture of the current state of research in the field of germline development. This not only enhances our collective understanding of this vital developmental process but also highlights existing gaps and challenges in the current body of knowledge. Identifying these areas is crucial as it sets the direction for future research endeavors, guiding scientists and researchers in their quest to unravel the complexities of germline development and its implications for reproductive health and ART. In Chapter 2 of my thesis, I focus on the development of a transcription factor (TF)-based system for the differentiation of PGCLCs. By inducing the overexpression of four key transcription factors—Prdm1, Prdm14, Tfap2c, and Nanog—I have established an efficient, cost-effective method for the scalable and simple differentiation of PGCLCs. This 4TF-induced system not only generates PGCLCs that show transcriptomic similarities to in vivo PGCs, but also parallels the characteristics of cytokine-induced PGCLCs. Another significant aspect of this work is the demonstration that PGCLC differentiation via this TF-based method can be achieved on a scalable level and sustained over prolonged cultures. Additionally, I have shown the direct differentiation of formative state embryonic stem cells (ESCs) into PGCLCs, further simplifying the PGCLC differentiation process. Overall, the work presented in this chapter establishes a powerful in vitro platform for germline modeling. In Chapter 3 of my thesis, I applied the TF-based system for PGCLC differentiation in combination with CRISPR inhibition (CRISPRi) technology to conduct a comprehensive screen for epigenetic regulators that influence PGC development. Through this approach, I targeted a total of 701 genes to identify those that significantly affect PGCLC formation. From this extensive screen, 53 genes were identified as key influencers in the efficiency of PGCLC formation. Notably, NCOR2, a transcriptional repressor known for its role in recruiting Class I and IIa histone deacetylases (HDACs) to target genes, emerged as an influential factor in inhibiting PGCLC differentiation. This finding aligns with existing evidence that underscores the importance of histone deacetylation in germline differentiation. Further, the study explored the impact of HDAC inhibitors (HDACi) on the differentiation process from ESCs to PGCLCs. Compounds like valproic acid and sodium butyrate were found to suppress this differentiation. Transcriptomic analysis of cultures treated with HDACi during ESC to PGCLC differentiation revealed a suppression of germline-associated pathways and an upregulation of somatic pathways. This work demonstrates the feasibility of conducting large scale screens of genes, chemical agents, and possibly genetic variants that impact germline development and epigenome.