Summary Macrophages are critical components of the innate immune system that sample the local environment, respond to stimuli and return tissues to homeostasis. Distinct macrophage populations play unique roles in these processes. Macrophages derived from circulating monocytes are rapidly recruited to infections, are inflammatory, and are generally short-lived. In contrast, long-lived tissue resident macrophages (TRMs) are derived from fetal liver cells and play an important role in maintaining homeostasis in the absence of infections. While monocyte-derived macrophages are well defined, due to experimental limitations there remain many open questions regarding how TRMs are maintained and contribute to regulating the local environment. To address these key gaps in knowledge my research group is developing new ex vivo models of distinct TRM populations that can then be probed using functional genetics. We recently developed an ex vivo model for lung-specific TRMs, alveolar macrophages (AMs), that maintain expression of AM-specific markers and function. Using this model we engineered a genome-wide knockout library in AM-like cells that enables rapid forward genetic screens using our customized screening pipeline. Over the next five years my research group will leverage this innovative resource to dissect underlying biological mechanisms related to AM maintenance and function. We will comprehensively define the genes required to maintain cells in the AM-like state using a combination of iterative genetic screens with in-depth functional characterization. These experiments will uncover entirely novel signaling and transcriptional networks activated in AMs during homeostasis. In parallel, we will compare the genetic control of core macrophage functions between myeloid-derived macrophages (BMDMs) and AM-like cells. While there are metabolic and transcriptional differences between BMDMs and AMs, it remains entirely unknown how these differences alter genetic control of macrophage functions like phagocytosis. We will complete screens in both BMDMs and AM-like cells probing phagocytosis of distinct cargo. These datasets will define shared and unique pathways that control core macrophage functions and will illuminate new mechanisms of fundamental biological processes. Finally, my research group will lay the groundwork for dissecting genetic pathways in AMs in intact animals by optimizing a cell transfer and screening pipeline. Using this model, we will uncover the in vivo role of key AM genes and identify new pathways required for AMs to maintain lung homeostasis. Accomplishing these goals will position my research group to understand the underlying mechanisms controlling AMs in detail not previously possible. Our long- term goal is to expand these approaches and findings to more broadly understand other TRM populations by identifying shared mechanisms of function and maintenance.