Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34(+) hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.