ABSTRACT The balance between hematopoietic stem cell (HSC) self-renewal and differentiation directly impacts hematopoietic homeostasis. We hypothesize that signals from the bone marrow microenvironment (or “niche”), together with cues from cell-intrinsic networks, contribute to fine-tuning this balance. However, our understanding of the niche has been limited by the current approach relying on sequential deletion of individual regulatory factors from candidate cells in available mouse models, and analysis of individual HSCs and their in vivo interactions with the niche has also been hindered by the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate in vivo. To illuminate the behavior of individual HSCs in vivo, we have established a new technical regimen which includes 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 marrow for transcriptomic assay. Our project will utilize these advances to describe the molecular basis of HSC fate choice in the niche. This in turn will facilitate novel therapeutic strategies for cell- fate manipulation which could accelerate hematopoietic recovery after transplantation, and possibly contribute to improved transplantation efficiency for non-malignant blood diseases. Thus, the goals of this proposal are three-fold: (1) to identify molecular mechanisms which enhance symmetric self-renewing division of HSCs, (2) to understand the niche factors governing HSC division balance, and (3) to assess the HSC niche under non- genotoxic conditioning. If successful, the proposed research will positively impact the HSC field by identifying molecular targets that will improve hematopoietic recovery after transplantation, and enable improvements in the ex vivo engineering of niche models.