Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (−)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562 cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (−). Furthermore, the number of K562 cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (−). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562 cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions in vitro. Display omitted •The autonomous hyperproliferation of K562 cells was suppressed in the 3D niche.•K562 cell differentiation was induced in the 3D niche despite the lack of arginine.•Increased K562 cells in the G0/G1 phase were observed in the 3D stromal niche.•Stroma in the 3D niche affects leukemic cell growth by controlling the cell cycle.