Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3(+) regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3(+) regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3(+) regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4(+) T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4(+) T cells are induced from naive CD4(+) T cells in response to antigenic stimulation. FoxP3(+) regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFbeta. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3(+) regulatory T cells. We conclude that FoxP3(+) T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4(+) T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.