PROJECT SUMMARY Emergency myelopoiesis (EM) is a rapid response by hematopoietic stem and progenitor cells (HSPCs) that is activated in response to inflammatory stimuli such as severe infection and cancer, wherein HSPCs proliferate and in increase their output of myeloid cells to replenish bone marrow reserves of those that have been deployed to sites of inflammation. Accumulating evidence has now shown that following the resolution of inflammation, HSPCs can maintain an altered epigenetic program over time. This process, termed “trained immunity” is a type of epigenetic memory that can result in more rapid responses to subsequent inflammatory challenges, such as infection and cancer. Despite growing evidence of its importance in regulating responses to inflammation, the cellular players and molecular pathways involved in controlling the epigenetic responses that lead to trained immunity are poorly understood. In preliminary studies, we performed scRNA-seq in sorted bone marrow immune cells following challenge with β-glucan, a stimulus which induces trained immunity following a rapid EM response. This analysis identified robust activation of bone marrow group 2 innate lymphoid cells (ILC2s) to produce GM-CSF. Blocking either GM-CSF or deleting ILC2s abrogated the EM response β-glucan. However, whether ILC2s or GM-CSF promote trained immunity in this model is not known. Based on this data, we propose that GM-CSF elaborated by ILC2s in response to β-glucan plays a critical role in the establishment of trained immunity. Here we propose to test this hypothesis by (i) defining the role of GM-CSF in trained immunity; (ii) establish the role of ILC2s in this system through an ILC2-deficient mouse line; and (iii) determining the gene expression networks and epigenetic landscapes established by GM-CSF downstream of β-glucan challenge in HSPCs. Altogether, completion of these studies will fundamentally advance our understanding of the regulation of trained immunity. Furthermore, as trained immunity is actively investigated as an intervention in the clinic, modulation of ILC2-GM-CSF pathway could be a novel therapeutic target for the treatment of inflammatory diseases and cancer. Finally, completion of these studies will establish the groundwork for future efforts to identify key cellular and molecular pathways involved in regulating trained immunity in vivo.