DNA methylation plays important roles in a wide range of biological phenomena, especially in the embryonic development and tumorigenesis. However, correlations between differentiation and DNA methylation have not been clarified well in each differentiation system. In this study, we focused our attention on regulatory roles of DNA methylation in normal hematopoietic differentiation using a demethylating reagent, 5-azacytidine (5-AzaC). As a source of hematopoietic progenitor cells, we used CD34(+) cells prepared from human umbilical cord blood and examined the effects of 5-AzaC on the colony-forming activity and the long-term culture-initiating (LTC-IC) activity of these cells. 5-AzaC treatment increased LTC-IC frequency 1.57- to 2.50-fold as compared to the nontreated control. In parallel to this, immunoblotting analysis showed that the intensity of overall DNA methylation decreased after 5-AzaC treatment. These results indicated the involvement of DNA methylation and demethylation in controlling immaturity of hematopoietic progenitor cells and the usefulness of 5-AzaC for regulating this immaturity.