Project Summary Hematopoiesis is the process in bone marrow by which all blood cells are generated throughout organismal life. Normal hematopoiesis is impaired in multiple diseases including anemias, cytopenias, and hematological malignancies such as leukemias and lymphomas. Little is known about the overall dynamics and progenitor hierarchy of steady-state unperturbed hematopoiesis (as opposed to the transplant setting), and how these are affected by oncogenic transformation or iatrogenic insults such as lineage ablation. Our overall goal is to characterize in detail the in situ dynamics of physiological hematopoiesis and progenitor phenotypes at steady- state and during adaptation to hematopoietic disruption. We propose to employ specific labeling and lineage tracing of hematopoietic stem cells (HSC) with cellular indexing of transcriptomes and epitopes by single cell sequencing (CITE-Seq) to accomplish three specific aims. The first is to characterize the intermediate progenitor phenotypes and dynamics in an unbiased manner during steady-state hematopoiesis. In doing so, we will construct a comprehensive and unbiased roadmap of hematopoietic differentiation. The second aim is to investigate adaptation of hematopoiesis to leukemogenic perturbation. And lastly, the third aim is to elucidate hematopoietic adaptation to the clinically relevant procedure of mature hematopoietic cell ablation. The proposed studies will describe for the first time the kinetics of hematopoietic differentiation in a variety of physiological, pathological and iatrogenic settings. To accomplish this goal, we will synergistically integrate cutting-edge techniques in an innovative way. These studies would significantly advance the field of hematology, paving the way for the development of novel therapies for maladies caused by disruption of normal hematopoietic differentiation.