Several studies have shown defective progenitor-stromal interactions in chronic myeloid leukemia (CML), and adhesive defects induced by BCR/ABL have been described. However, controversial results have been reported, and the role of the stroma in abnormal development of the hematopoietic system is not clear. In this study, CML hematopoietic and irradiated stromal cells were co-cultured in different combinations for 10 or 21 days. Maintenance of viable cells was dependent both on the sources of hematopoietic progenitors and stromal adherent layers, with normal cells performing better than their leukemic counterparts. The frequency of CD34(+) CD38(-) cells in the non-adherent fraction was more related to the source of hematopoietic cells than of stroma, and hematopoietic cells from normal subjects showed better performance. The simultaneous analysis of different combinations of normal and leukemic precursor cells and stromal layers, as done in the present work, suggests that the outcome of the interaction depends on characteristics of both compartments. This hematopoietic system development is influenced by intrinsic qualities of both hematopoietic stem cells and the supportive stroma.