Granulocyte colony-stimulating factor (G-CSF) is routinely used to collect peripheral blood stem cells (PBSCs) from healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT). We show that, in both humans and mice, G-CSF mobilizes a subset of CD34+ cells with mature monocyte features. These cells, which are phenotypically and functionally conserved in mice and humans, are transcriptionally distinct from myeloid and monocytic precursors but similar to mature monocytes and endowed with immunosuppressive properties. In response to interferon-γ released by activated T cells, these cells produce nitric oxide, which induces allogeneic T cell death both in vitro and in vivo. These apoptotic T cells are engulfed by macrophages that release transforming growth factor–β and promote regulatory T cell expansion. Indeed, the fraction of CD34+ monocytes in peripheral blood CD34+ cells inversely correlates with the incidence of acute graft-versus-host disease (GVHD) in humans. Therefore, G-CSF–mobilized cells are an attractive candidate population to be expanded ex vivo for cellular therapy against GVHD.