Recent technological advancements and genome-wide studies provide compelling evidence that dynamic chromatin interaction and three-dimensional genome organization in nuclei play an important role in regulating gene expression. Mammalian genomes consist of many small functional domains termed topologically associated domains (TADs), many of them organized by CCCTC-binding factor (CTCF) and the cohesion complex. Changes in genome TADs might result in inappropriate promoter/enhancer communications leading to activation of oncogenes or suppression of tumor suppressors. During normal hematopoiesis and leukemogenesis, genome structure alters considerably to facilitate normal and malignant hematopoiesis, respectively. Delineating theses normal and abnormal processes will evolve our understanding of disease pathogenesis and development of potential treatment strategies. This review highlights the role of CTCF and its associated protein complexes in three-dimensional genome organization in development and leukemogenesis, as well as the roles of CTCF boundary defined TAD in transcription regulation. We further explore the function of chromatin modulators, such as CTCF, cohesin, and long noncoding RNAs (lncRNAs) in chromosomal interactions and hematopoietic genome organization. Finally, we focus on the implication of 3D genome alteration in the pathogenesis of leukemia and provide a scientific basis for targeted intervention.