Abstract: c-myb is an important transcription factor in the process of hematopoiesis and is related to the proliferation, differentiation and apoptosis of hematopoietic stem cells. In malignant tumors such as leukemia, colon cancer, breast cancer and melanoma, c-myb is abnormally expressed, but the mechanism of c-myb regulation in leukemia cells is unclear. This study explored the relationship between GATA1 and c-myb in U937 cells. Regulating the relationship may help leukemia research and treatment. The differentiation of U937 cells was induced with12-O-tetradecanoylphorbol-13-acetate（TPA）, and the expressions of GATA1 and c-myb protein before and after differentiation were detected. Western blot results showed that c-myb and GATA1 protein decreased significantly after U937 cell differentiation induced by TPA. After lentivirus packaging GATA1 shRNA plasmid and GATA1 overexpression plasmid and infecting U937 cells to achieve knockdown and overexpression of transcription factor GATA1, the expression levels of c-myb mRNA and protein were detected. The results showed that after knocking down GATA1, the mRNA and protein levels of c-myb decreased significantly; after overexpressing GATA1, the m RNA and protein levels of c-myb increased significantly. Our research revealed the positive regulation of c-myb by GATA1 in U937 cells, and provided new ideas for leukemia research and treatment options.