The differentiation of eosinophils from hematopoietic precursors and their subsequent maturation, chemotaxis, and activation is primarily regulated by interleukin-5 (IL-5). To examine the effect of chronic IL-5 exposure on hematopoiesis, IL-5 transgenic (IL-5trg) mice and wild-type BALB/c (WT) mice were examined. In comparison to WT mice, a significant alteration in bone marrow hematopoiesis was observed in IL-5trg mice. Although the total number of myeloid progenitors in the bone marrow of IL-5trg mice was not significantly altered, the number of long-term culture-initiating cells (LTC-ICs) was 1.5-fold lower than that observed in WT mice. Furthermore, IL-5trg mice failed to demonstrate hematopoietic activity in long-term bone marrow cultures, which correlated with a significant decrease in the number of bone marrow mesenchymal/stromal progenitor (MSP) cells in these mice. In comparison to WT mice, a 10-fold decrease was observed in the number of fibroblast colony-forming units (CFU-Fs) in IL-5trg bone marrow. Hematopoietic activity of IL-5trg bone marrow cells was rescued by cultivation on preestablished layers of bone marrow-derived stromal cells. However, in contrast to bone marrow, increased hematopoietic activity was observed in the spleen and peripheral blood of IL-5trg mice. Likewise, the numbers of LTC-ICs and granulocyte-macrophage, macrophage, eosinophil, B-lymphocyte progenitors in the peripheral blood and spleen of IL-5trg mice were approximately 20-fold higher than in WT mice. A significant increase in CFU-F numbers was also observed in the spleens of IL-5trg mice compared with WT mice. Overall, our results suggest that constitutive overexpression of IL-5 can potentially induce colonization of spleen with MSP cells, which provides the necessary microenvironment for establishment of hematopoiesis in extramedullary sites.