ABSTRACT The symmetry of stem cell division is one of the most fundamental questions in stem cell biology, and a leading goal of our research is identification of the key metabolic pathways that regulate hematopoietic stem cell (HSC) fate. We hypothesize that lipid metabolism contributes to HSC maintenance through precise control of division patterns. Single-cell approaches have identified the enhanced clearance of damaged mitochondria by fatty acid oxidation as an important mechanism of the self-renewing expansion of HSCs. However, our understanding of the relationship between HSC self-renewal and lipid metabolism is limited, as analyses of individual HSC division patterns have been hindered by both the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate in vivo. In addition, the number of cells required for full metabolomics analysis of rare populations of HSCs has proven prohibitive. To examine the activity upstream of fatty acid oxidation in HSCs, we have generated hematopoietic-specific conditional knockout mice for key genes impacting fatty acid oxidation pathway and/or fatty acid flow. A new biosensor for assessment of fatty acid oxidation activity in live cells has likewise been established to determine the metabolic modes which are most relevant to the controlled equilibrium of HSCs, and the gene-expression oriented bioinformatics tool, graphite, has been adapted to identify specific metabolite-dependent pathways. In order to illuminate the behavior of individual HSCs in vivo, we have established new technical regimens which include prospective isolation of HSCs with high purity based on Tie2 positivity, a local transplantation technique which delivers a single HSC under multiphoton microscopy guidance into the bone marrow of a live mouse, and micropipette aspiration to extract single cells after division directly from the bone marrow for functional or transcriptomic assay. Our project will utilize these advances to test our hypothesis regarding the roles of lipid metabolism in HSC fate choice. This in turn will facilitate novel therapeutic strategies for shifting the division balance of HSCs toward self-renewal through metabolic manipulation, and possibly contribute to improved clinical outcomes after HSC transplantation for non-malignant blood diseases. Thus, the goals of this proposal are three-fold: (1) In Aim 1, we will investigate the function of mitochondrial fatty acid oxidation in HSC division symmetry and explore a potential source of fatty acids to fulfill the requirements of HSCs; (2) In Aim 2, we will use the biosensor to identify key downstream metabolic targets of fatty acid metabolism for HSC fate and explore the measurement of the cellular metabolome in HSCs; and (3) finally, we propose in Aim 3 to directly examine in vivo HSC division symmetry, and the resulting division balance of fatty acid oxidation-defective HSCs will show definitively the in vivo relevance of fatty acid metabolism to HSC fate. If successful, the proposed research will positively impact the HSC field by providing a deeper understanding of the metabolic cues governing HSC fate decisions.