Cell fractionation techniques have been used for the purification and characterization of hematopoietic cells present in peripheral blood and bone marrow. Following fractionation, the distribution of hematopoietic cells is frequently determined by the granulocyte-monocyte colony-forming unit (CFU-gm) assay. In this study, we questioned whether the purification process itself altered the sensitivity of the CFU-gm assay through changes in the accessory cell populations. Experiments showed that following T cell depletion, the cloning efficiency of CFU-gm was suboptimal, since the addition of autologous T lymphocytes was stimulatory, even when the concentration of conditioned medium was optimal. In contrast, growth of CFU-gm was inhibited by monocytes, both in the presence and absence of T cells. Sensitivity to monocyte-derived inhibition occurred at significantly lower monocyte concentrations when T cells were present. Thus, stem cell purification techniques which deplete T lymphocytes or enrich monocytes have an adverse effect on the cloning efficiency of peripheral blood CFU-gm. This study presents culture techniques capable of circumventing these problems.