ABSTRACT Type 3 lymphocytes include group 3 innate lymphoid cells (ILC3), IL-17-producing γδ T cells (Tγδ17), and T helper 17 ( TH17) cells and are crucial for maintaining mucosal barrier defenses against extracellular bacteria and fungi. Nonetheless, dysregulated type 3 lymphocyte responses have been implicated in the pathogenesis of several autoimmune diseases, such as inflammatory bowel disease, Crohn's disease, and multiple sclerosis. ILC3, Tγδ17, and TH17 cells are defined by their shared expression of the lineage-defining transcription factor RORγt (Rorc(t)), which facilitates key inductive events during the development and differentiation of these cell types towards the type 3 lymphocyte lineage. The trans-acting factor networks that cooperatively regulate the formation of the type 3 lymphocyte cell types have been well characterized, however, the cis-regulatory elements (CREs) that control the development and differentiation of type 3 lymphocytes remain less defined Therefore, we are proposing a comprehensive assessment of the CREs controlling the expression of the Rorc(t) locus across the type 3 lymphocyte lineage cell types. To this end, we will (i) identify the CREs governing Rorc(t) expression using a high-throughput CRISPRi knockdown screen; (ii) evaluate the activity of Rorc(t) CREs using a retroviral massively parallel STARR-seq reporter assay; (iii) and determine the functional role of putative CREs for the expression of Rorc(t) in the ILC3, Tγδ17, and TH17 cell types. Altogether, the experiments outlined in this proposal will expand our understanding of the cis-regulatory mechanisms governing type 3 lymphocyte development and identity.