Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis. In particular, the effects of hydrogen peroxide (H2O2) range from hematopoietic cell proliferation to cell death, depending on its concentration in the intracellular milieu. In this work, we evaluated the effects of an oxidative environment on normal and leukemic hematopoietic cells by stimulating normal human (umbilical cord blood) and murine (bone marrow) hematopoietic cells, as well as human myeloid leukemic cells (HL-60 lineage), upon H2O2 stimulus. Total cell populations and primitive subsets were evaluated for each cell type. H2O2 stimulus induces HL-60 cell death, whereas the viability of human and murine normal cells was not affected. The effects of H2O2 stimulus on hematopoietic stem/progenitor cell subsets were examined and the normal primitive cells were found to be unaffected; however, the percentage of leukemic stem cells (LSC) increased in response to H2O2, while clonogenic ability of these cells to generate myeloid clones was inhibited. In addition, H2O2 stimulus caused a decrease in the levels of p-AKT in HL-60 cells, which most likely mediates the observed decrease of viability. In summary, we found that at low concentrations, H2O2 preferentially affects both the LSC subset and total HL-60 cells without damage normal cells. © 2013 Nogueira-Pedro et al.; licensee BioMed Central Ltd.