Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM(5). Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis. Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency. Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC) and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.