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Granulocyte colony-stimulating factor (G-CSF) mobilizes regulatory T cells (Tregs) from bone marrow into the peripheral blood, by reducing the expression of stromal cell-derived factor-1α (SDF-1α). However, G-CSF has rarely been studied in acute myeloid leukemia (AML) immunotherapy. The present study performed a Transwell migration assay in vitro to determine the contribution of SDF-1α to the migration of leukemia cells, and the effects of G-CSF were evaluated. The effects of G-CSF on SDF-1α and Tregs in the AML microenvironment were examined, by employing a WEHI-3-grafted BALB/c mouse AML model (AML-M4). It is evident that G-CSF reversed immunosuppression of the AML microenvironment by reducing SDF-1α in bone marrow and elevating Tregs in the peripheral blood in in vivo studies. Furthermore, AML mice treated with vaccines combined with G-CSF achieved a longer survival time than those treated with vaccines without G-CSF, showing the efficiency of the regimen. The present study demonstrates the effects of G-CSF on the mobilization of leukemia cells and Tregs into the peripheral blood. In addition, immunotherapy with G-CSF priming represents a promising therapeutic strategy of targeting the immunosuppression.