M cells have been shown to sample luminal antigens and use transcytosis to deliver these antigens to underlying antigen presenting dendritic cells. Thus, they serve as key regulators of mucosal immunity. Despite the vast body of literature on the immunologic role of M cells in other mucosal organs, airway M cells have only recently been described and almost no functional or molecular characterization has been performed. We present the first single cell transcriptomes of murine airway M cells and identify airway M cell signaling cascades that regulate inflammation. We have discovered that airway M cells occur as solitary cells in the murine trachea and as patches in the murine small airway, and we report the directed differentiation of human airway M cells from primary human airway epithelium. Finally, we demonstrate that airway M cells are induced by the administration of the signaling factor RANKL, treatment with Lipopolysaccharide (LPS), and influenza infection. Interestingly, M cells can occur as solitary cells, but in the setting of physiologic stimuli like LPS or influenza infection, patches of M cells are associated with lymphoid follicles suggesting a functional epithelial-immune unit. In this grant application, we propose to use our combined expertise in stem cell biology, epithelial biology, lung inflammation, and influenza infection to define the functional biology of lung M cells. We will start by defining the cellular origins of murine and human airway M cells. We have now generated a comprehensive battery of murine genetic driver lines for lineage tracing all the cells of airway epithelium. Herein, we propose to deploy this set of murine lines, for the first time, in order to precisely define all the putative parental epithelial cell types that can give rise to M cells. We will also clarify the role of RANKL-RANK signaling pathways in murine models of inflammation. Finally, to define airway M cell functions, we constructed a new Sox8-CreER driver mouse that allows us to specifically label, genetically modify, and ablate M cells. We will integrate these genetic reagents with existing murine disease models to elucidate the role of functional roles of M cells in models of airway inflammation and influenza infection. Lastly, we will dissect the molecular mechanisms of two candidate chemokines that govern M cell-immune cell interactions using genetic and pharmacological manipulation in combination with a novel tracheal explant and lung slice live imaging platform.