PROJECT SUMMARY Hematopoietic Stem Cells (HSCs) are clinically valuable because they display regenerative properties of self- renewal and the ability to produce an entire hematopoietic system through multilineage differentiation. In other words, HSC can make all mature blood cell types such as myeloid, erythroid, and lymphoid and they sustain those blood types long-term in the organism. These regenerative properties make them an idea therapeutic tool for treating hematological malignancies by transplantation from a healthy donor, however a shortage of donors has prompted the effort to generate HSCs from alternative sources such as patient derived iPSCs. Knowledge about molecular mechanisms of HSC formation is needed to aid those efforts. HSCs are formed in early embryonic development alongside other HSC-independent Progenitor Cells (HPCs). One major difference between them is that HPCs are more limited in their differentiation repertoire compared to HSCs. We aim to study the differences between HSCs and HPCs to further advance our understanding of the specific molecular drivers of HSC regenerative properties. Understanding how HSCs acquire the ability to differentiate and self- renew in development can aid the efforts for generating them in vitro from alternative sources such as patient derived tissues or cord blood sources. Current molecular markers are insufficient for distinguishing HSCs from all other HPC subtypes. Moreover, most experimental systems require the removal of these cells from their native environment to study their functionality using in vitro culture assays or transplantation, which will measure the potential of a cell but may not reflect its actual endogenous function. Genetic experiments in mice and zebrafish showed that HSC and HPC fate can be molecularly uncoupled, implying that unique drivers of HSC formation exist. What genes are responsible for the formation of the rare population of HSCs independently from HPCs? To address this question, we established a genetic lineage tracing approach that measures HSC and HPC differentiation without removing them from their native environment. Using this assay, we found that HSCs show a significant delay in their ability to differentiate to myeloid and lymphoid lineages relative to HPCs, and this finding agrees with others in the field. The assay allows us to assess HSC differentiation in vivo, uncoupled from HPCs. We propose to use this functional tool in combination with other molecular and genetic assays to address the following aims: (1) Determine the differentiation potential of HSCs and HPCs in vivo and (2) Perform a reverse genetic screen for regulators of HSC differentiation. Our findings can aid the efforts to generate HSCs from alternative sources for clinical use.