Chronic human exposure to benzene has been linked to several hematopoietic disorders, including leukemia and lymphomas. Certain benzene metabolites, including benzoquinone (BQ), are genotoxic and mutagenic. Bone marrow stem cells are targets for benzene-induced cytotoxicity and DNA damage that could result in changes to the genome of these progenitor cells, thereby leading to hematopoietic disorders and cancers. Human bone marrow CD34+ hematopoietic progenitor cells (HPC) were exposed in vitro to 1,4-BQ to assess cytotoxicity, genotoxicity, and DNA damage responses and the molecular mechanisms associated with these events. CD34+ HPC from 10 men and 10 women were exposed to 0, 1, 5, 10, 15, or 20 μM of 1,4-BQ and analyzed 72 h later. Apoptosis and cytotoxicity were dose-dependent, with exposure to 10 μM 1,4-BQ resulting in approximately 60% cytotoxicity relative to untreated controls. A significant increase in the percentage of micronucleated CD34+ cells was detected in cultures treated with 1,4-BQ. In addition, the p21 mRNA level was elevated in 1,4-BQ-treated cells, suggesting that human CD34+ cells utilize the p53 pathway in response to 1,4-BQ-induced DNA damage. However, there were no significant changes in mRNA levels of the DNA repair genes ku80, rad51, xpa, xpc, and ape1 as well as p53 following treatment with 1,4-BQ. Although interindividual variations were evident in the cellular response to 1,4-BQ, there was no gender difference in the response overall. These results show that human CD34+ cells are sensitive targets for 1,4-BQ toxicity that use the p53 DNA damage response pathway in response to genotoxic stress. Human CD34+ HPC will be useful for testing the toxicity of other benzene metabolites and various hematotoxic chemicals.