Introduction: Benzene is commonly blamed to suppress hematopoiesis in humans and experimental animals by either toxic or suppressive mechanisms. We hypothesized that interferon-γ, representing a key mediator in immune mediated Aplastic Anemia (AA) also plays a role in benzene-mediated hematopoietic dysfunction.Material and Methods: C57B1/6 mice were s.c. immunized with either 100 nM benzene, 100 nM benzoquinone (BQ) or 100 nM hydroquinone (HQ) in PBS/ethanol. Five mice were in each setting. Control mice received PBS/ethanol alone. On day 2, the draining lymphnodes were minced, fixed and stained for cytoplasmic IFN-γ using a FITC-labeled monoclonal antibody. Results are displayed as dot plots of the lymphocyte gate after flow cytometry.Results: Benzene, hydroquinone but not benzoquinone induce the accumulation of interferon-γ secreting cells in the popliteal lymphnode (PLN) on day 2 after immunization (HQ 100 nM/mouse; mean: 2.47% ± 0.91 IFN-γ positive cells; benzene 100 nM/mouse; mean: 2.14% ± 0.06 IFN-γ positive cells; BQ 100 nM/mouse; mean: 0.11% ± 0.09 IFN-γ positive cells). Concomitantly, we found an increase of heat shock protein 70 positive cells (HQ > benzene ≥ BQ). The peak of IFN-γ positive cells on day 2 did not result in a significant increase of cell counts in the popliteal lymphnode assay. By contrast, significant lymphnode enlargement was attained on day 6. Out of all metabolites analyzed, the highest PLN-index was detected after benzoquinone immunization (100 nM BQ; PLN-index: 15.12 ± 3.72) followed by hydroquinone (100 nM HQ; PLN-index: 3.64 ± 1.25) and almost no cellcount increases occurred after benzene immunization.Conclusions: Low doses of hydroquinone, as well as benzene induce interferon-γ secreting cells in draining lymphnodes of C57B1/6 mice. Interferon-γ could play an important role in stimulating antigen presenting cell (APC) activation in the absence of specific antigen. Benzoquinone, a metabolite of benzene, could be responsible for neoantigen formation and both metabolites, BQ and HQ, might explain the hyperproliferation of T cells. Uncontrolled hyperproliferation of lymphocytes in the hematopoietic microenvironment could be responsible for the accumulation of suppressive mediators.