Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34(+) cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34(+) cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell-stromal cell interactions.