Abstract The mechanism by which stem and progenitor cells commit to proliferate, self-renew, or differentiate towards different lineages remains unclear. Understanding this process is essential to decipher both normal tissue development and aberrant differentiation associated with malignant transformation. CUX1 is a non-clustered HOX-family transcription factor that is recurrently mutated in hematopoietic and solid tumours and regulates cell fate in multiple developmental systems. We reported that CUX1 is required for normal hematopoietic homeostasis of both murine and human hematopoietic stem and progenitor cells (HSPCs). To date, the transcriptional role of CUX1 in HSPCs has been unclear, partly because of the complexity of the Cux1 locus. Namely, Cux1 shares exons with a distinct gene, Casp, which encodes a highly expressed golgi-associated protein. As most transcriptomic studies combine Casp and Cux1 transcripts, the nuances of Cux1 expression are concealed. To identify the transcriptional mechanism by which CUX1 regulates HSPC functions, we compared scRNA-seq data of Kit+lin- HSPCs from transgenic mice expressing inducible shRNAs targeting Cux1, Cux1mid and Cux1low, to control counterparts expressing an shRNA targeting renilla luciferase. We demonstrate that separating Cux1 from Casp transcripts provides more power to detect the influence of Cux1 in scRNA-seq data analysis. While cell numbers within most HSPC sub-populations are largely unchanged after five days of Cux1 knockdown, the transcriptomes within multiple clusters are disrupted. The effect size is greatest in Cux1low cells, compared to Cux1mid, consistent with dosage dependent roles of CUX1 in HSPCs. Across sub-populations, Cux1 levels correlated with gene signatures of “stemness,” and Cux1 knockdown disrupted this gene signature in the core hematopoietic stem cell (HSC) population. Concomitantly, low levels of Cux1 led to upregulation of cellular metabolism and G2/M cell cycle transition pathways in core HSCs, indicating that CUX1 regulates stem cell quiescence at the hematopoietic apex. We are currently integrating this data with epigenetic datasets to determine the mechanism by which CUX1 regulates critical hematopoietic transcriptional programs. Overall, understanding the regulatory function of CUX1 will provide insight into how HSCs commit to different fates and may ultimately lead to novel cancer therapeutic interventions. Citation Format: Weihan Liu, Stephanie Konecki, Tanner Martinez, Megan McNerney. CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3159.