Hematopoietic stem cells allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. Hematopoietic cell fate decisions such as self‐renewal and differentiation are highly regulated through multiple molecular pathways and dysregulation can lead to the initiation of malignant hematopoietic disorders like acute myeloid leukemia (AML), immune deficiencies, and anemia. One pathway that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component, the protein that decides which substrate will be chosen for degradation, of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21was highly expressed in the hematopoietic stem and progenitor (HSPC) population, and was more highly expressed in this population when compared to other Fbox genes suggesting a possible role in maintaining the HSPC population. Likewise, Western blot confirmed high levels of FBXO21 in HSPCs and revealed low to no protein in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self‐renewal, and differentiation, we generated shRNAs against both Fbxo21 and FBXO21. We found that silencing Fbxo21/FBXO21 in healthy HSPCs and in human AML cell lines lead to a loss in colony formation, a decrease in proliferation, and an increase in differentiation towards the mature myeloid lineage. While overexpression of FBXO21 lead to an increase in colony formation and proliferation. This leads us to hypothesize that the ubiquitin E3 ligase, FBXO21, maintains the hematopoietic stem cell population while loss of Fbxo21/FBXO21 leads to a promotion in myeloid differentiation.We then generated the first conditional Fbxo21 knockout (KO) mouse model to delete Fbxo21 in hematopoietic cells. Our Fbxo21 KO mice exhibited no significant difference in primary sites of hematopoietic development, but display changes in hematopoietic cell populations in both young and aged mice. To further identify the substrates and pathway of FBXO21, we performed TMT Mass Spectrometry to analyze changes in protein levels upon knockdown of Fbxo21/FBXO21 in healthy HSPC and human AML cell lines. We found that both populations displayed an increase in proteins involved in the inflammatory response, the immune response primarily maintained by the myeloid cells through cytokine signaling. Which among both our Fbxo21 KO mice and human AML cell lines with FBXO21 knocked down, we see alterations in the cell’s response to certain cytokines, including IL‐6 and G‐CSF, upon Fbxo21/FBXO21 being knocked down. Therefore, deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

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