Maintaining and restoring hematopoietic homeostasis is necessary to respond to hematopoietic stressors throughout our lifetime; the inability to do so can lead individuals to succumb to deadly hematopoietic threats like infection, hemorrhage, or anemia. Anemia is the most common blood disorder and is caused by deficient or dysfunctional red blood cells (RBCs). Previous work demonstrated that lipids, such as cholesterol, and their synthesis and/or metabolism contribute to erythropoiesis. However, the cholesterol synthesis pathway (CSP) has yet to be investigated as a targetable pathway to correct red blood cell disorders. Thus, there is an unmet medical need to mechanistically interrogate the role of lipids and their metabolism in regulating erythropoiesis, which this proposal seeks to fill. The CSP is an enzyme-driven process that generates cholesterol and isoprenoids. Mutations to the CSP give rise to anemia, but how the CSP regulates erythropoiesis remains to be defined. Our preliminary identified a role for the CSP in regulating RBC production and functions, but whether this occurs intrinsically or extrinsically is unclear. The objective of this proposal is to elucidate the function of distinct enzymes in the CSP in RBC production. The rationale underlying this proposal is that understanding the fundamental role of the CSP in RBC homeostasis is likely to identify novel targets that could be leveraged to treat patients with red blood cell disorders. We will test our central hypothesis that the CSP regulates erythropoiesis via alterations in hematopoietic cell proliferation and death. In Specific Aim 1, we will determine how the CSP controls erythropoiesis through erythroid progenitor cells. In Specific Aim 2, we will examine the function of hematopoietic stem cell CSP in regulating hematopoietic cell fate and self-renewal. Therefore, understanding how RBC homeostasis is regulated is likely to identify novel targets that could be used to accelerate hematologic recovery from stress.