The human leukocyte antigen-G (HLA-G) has been considered to be an important tolerogeneic molecule playing an essential role in maternal-fetal tolerance, which constitutes the perfect example of successful physiological immunotolerance of semi-allografts. In this context, we investigated the putative role of this molecule in the allogeneic hematopoietic cell transplantation setting. Design and Methods The percentage of HLA-G + cells in peripheral blood of healthy donors and allo-transplanted patients was evaluated by flow cytometry. Their immunoregulatory and tolerogeneic properties were investigated in in vitro immunostimulatory and immunosuppression assays. Immunohistochemical analysis for HLA-G expression was performed in skin biopsies from allo-transplanted patients and correlated with the occurrence of graft-versus-host disease. Results We identified a CD14 +HLA-G pos population with an HLA-DR low phenotype and decreased in vitro immunostimulatory capacity circulating in peripheral blood of healthy individuals. Naturally occurring CD14 +HLA-G pos cells suppressed T-cell responses and exerted an immunotolerogenic action on T cells by rendering them hyporesponsive and immunosuppressive in vitro. After allogeneic hematopoietic cell transplantation, HLA-G pos cells increase in blood. Interestingly, besides an increase in CD14 +HLA-G pos cells, there was also a pronounced expansion of CD3 +HLA-G pos cells. Of note, CD3 +HLA-G pos and CD14 +HLA-G pos cells from transplanted patients were suppressive in in vitro lymphoproliferation assays. Furthermore, we found an upregulation of HLA-G expression in skin specimens from transplanted patients that correlated with graft-versus-host disease. Inflammatory cells infiltrating the dermis of transplanted patients were also HLA-G pos. Conclusions We report the presence of naturally occurring HLA-G pos monocytic cells with in vitro suppressive properties. HLA-G expressing regulatory blood cells were found in increased numbers after allogeneic transplantation. Epithelial cells in skin affected by graft-versus-host disease revealed elevated HLA-G expression.