This three-parts thesis presents discovery of the molecular mechanisms underpinning hematopoiesis and its dysregulation in cancer. Identifying tumor suppressor genes on chromosome regions affected by aneuploidy has been historically challenging due to the large number of genes involved. The first part of the thesis leveraged published genome-wide perturbation screen data and advancement in machine learning algorithms in recent years. This work led to a supervised machine learning workflow that systemically predicted the tumor suppressor gene-like activities for all chromosome 7 genes. The second and third parts focus on the multifaceted roles of CUX1 as a pioneer transcription factor. CUX1 is a homeodomain-containing transcription factor (TF) that is essential for development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanisms by which CUX1 regulates gene expression and differentiation remain poorly understood, creating a barrier to understanding the tumor suppressive functions of CUX1. Herein, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase DNA accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. Moreover, the thesis illuminates the intricate relationship between CUX1 and GATA1, two key regulators in erythropoiesis. In erythroid differentiation, CUX1 dynamically shifts binding targets from hematopoietic stem cell (HSC) -specific enhancers to erythroid specific enhancers co-bound by GATA1. CUX1 gatekeeps GATA1 from abnormal and promiscuous binding by direct physical shielding and indirect mechanisms. These data indicate that CUX1 possesses pioneer factor activities to epigenetically regulate hematopoietic lineage commitment and homeostasis. CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation. In the erythroid branch of hematopoiesis, CUX1 promotes healthy differentiation through ensuring proper GATA1 binding. By bridging molecular insights from aneuploidy, CUX1 epigenetic regulatory mechanisms, and CUX1-GATA1 interaction, this thesis provides novel insights of the molecular machinery governing hematopoiesis and offers novel perspectives on cancer biology and treatment strategies.