ABSTRACT During a recent single cell RNA sequencing analysis of the developing human lung, we identified a cell population characterized by a unique gene expression profile that has not previously been reported in the human or mouse lung. These cells express SCGB3A2/SFTPB/CFTR, but lack markers for other well characterized cell types such as club cells (SCGB1A1) or ionocytes (FOXI1). We refer to these as Fetal Airway Secretory (FAS) cells given the expression of several secretory genes within the transcriptional signature. Since nothing is known about FAS cells, and there is no analogous population in the murine lung, the overarching goal of this proposal is to both describe these cells in detail, to interrogate their differentiation potential, and to determine how these cells are regulated. By using an in vitro model of the developing human lung epithelium called human Bud Tip Progenitor (BTP)-organoids, preliminary data shows that BTP-organoids can give rise to both TP63+ progenitors and FAS cells during airway differentiation, and that this differentiation can be modulated by TGFb/BMP/SMAD signaling. Single cell barcode-based lineage tracing during the transition from BTP-to-airway suggest that clones of FAS cells or basal cells give rise to distinct subsets of airway cells, with FAS cells giving rise to pulmonary neuroendocrine cells (PNECs) and a subset of C6+ multiciliated (MC) cells, while TP63+ progenitor cells give rise to secretory cells and MUC16+ MC cells. Based on this data, we will test the overarching hypothesis that FAS cells differentiate distinct airway progenitor cell that give rise to a subset of airway cell types. Given the novelty of this cell type, and the unique tools that we have developed to assess the differentiating human epithelium in vitro, understanding this cell population will provide foundational descriptive and mechanistic insights into human lung biology.