Granulocyte colony‐stimulating factor (G‐CSF)‐mobilized donor graft tissue used for peripheral blood stem cell transplantation contains a large number of immature myeloid cells that suppress alloreactive donor T cells, resulting in an inhibition of acute graft‐versus‐host disease (GVHD). However, the molecular mechanism underlying the suppressive function of immature myeloid cells is not fully understood. Here, we investigated whether indoleamine 2,3‐dioxygenase (IDO) is related to the suppressive mechanism of G‐CSF‐induced immature myeloid cells (gMCs). We found that Gr‐1⁺ CD11b⁺ cells were highly induced in G‐CSF‐injected donor graft tissue, which is a phenotype of immature myeloid cells, resulting in an inhibition of acute GVHD lethality by suppressing alloreactive donor T‐cell expansion. IDO was not detected in primary isolated gMCs; however, this enzyme was markedly induced after treatment with interferon‐γ (IFN‐γ). This level was significantly higher in IFN‐γ‐treated gMCs than in bone marrow myeloid cells, which promote alloreactive T‐cell responses. We next investigated the functional role of IDO in gMC‐mediated inhibition of acute GVHD lethality. We found no changes in gMC‐mediated survival or alloreactive donor T‐cell suppression when IDO activity was blocked using 1‐methyl tryptophan. In addition, there was no difference in gMC‐mediated survival rates between recipients transferred with either wild‐type gMCs or IDO^−/− gMCs. Taken together, our data suggest that gMC‐mediated inhibition of lethal acute GVHD is through an IDO‐independent mechanism.