Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.