Abstract The overall aim of this proposal is to bring together the respective expertise of the Elliott and Palis labs in macrophage and erythroid biology, respectively, to better understand the role of tissue-resident macrophages in the regulation red blood cell production. While great progress has been made recently in understanding macrophage heterogeneity and tissue-specific function in many organs, including brain, skin, lungs, intestines, liver, and spleen, comparatively little is known about the diversity of macrophages in the bone marrow, where macrophages provide the microenvironmental niche for maturing erythroid precursors within ?erythroblastic islands?. In Aim 1, we will employ multidimensional flow cytometric approaches with functional tests to better define the identity of erythroid-associated macrophages (EA-Macs) in the bone marrow. Adult humans synthesize 2.5 million new red blood cells every second to maintain our circulating red cell mass, which constitutes >80% of all the cells in the body. Terminal erythroblasts in mammals enucleate to yield reticulocytes and pyrenocytes. An important function of EA-Macs is pyrenocyte clearance. In Aim 2 we will test the function of CD47 ?don't eat me? signals in pyrenocyte clearance, as well as the role of erythropoietin, the primary regulator of red cell production, in regulating the capacity of EA-Macs to clear pyrenocytes. Erythropoietin promotes the survival of late stage erythroid progenitors and immature erythroblasts, which together constitute the erythropoietin-responsive compartment of the erythron. Our preliminary studies in two independent- radiation and phlebotomy- models of stress erythropoiesis indicate that erythropoietin expands the erythropoietin-responsive compartment in the bone marrow in a macrophage-dependent manner. In Aim 3, we will test the novel hypothesis that EA-Macs constitute a critical component of the erythropoietin-responsive compartment during the recovery from acute anemia. Taken together these studies will establish fundamental insights regarding the microenvironmental regulation of the erythron by EA-Macs in the bone marrow and will lay the groundwork for the future study of the role of EA-Macs in erythroid-intrinsic diseases.