Acute graft-versus-host disease (aGVHD) represents the rejection of the recipient's skin, gut, and liver tissues of an allogeneic hematopoietic stem cell transplantation (HSCT) by the donor T cells. The onset of aGVHD is often rapid and its evolution is unpredictable. We undertook the single-cell RNA sequencing of peripheral blood mononuclear cells collected before aGVHD clinical onset in 3 patients and from 1 patient afterward. We used 4 HSCT recipients who remained free of aGVHD as controls. This analysis unveiled the presence of particular subpopulations of circulating monocytes and cytotoxic T cells (CTLs) in pre-aGVHD samples up to 18 d before clinical disease. These pre-aGVHD monocytes were characterized by an upregulation of the M2 polarity marker CD163 and the transmembrane protein SIGLEC1/CD169. At the same time, their CTL counterparts stood out for the upregulation of the CXCL10 receptor CXCR3 and the antigenic stimulation marker CD70. The occurrence of CD163/SIGLEC1 co-expressing monocytes upstream of aGVHD onset was validated using transcriptomic data from an independent cohort and by flow cytometry in additional blood samples. These findings point to potential early diagnostic tools and preventive therapeutic strategies for aGVHD.