Abstract Graft-versus-host disease (GvHD) is a severe complication of hematopoietic stem cell transplantation driven by activated allogeneic T cells. Here, we identify a distinct subset of T cell factor-1 (TCF1) + CD8 + T cells in mouse allogeneic and xenogeneic transplant models of acute GvHD. These TCF1 + cells exhibit distinct characteristics compared to TCF1 - cells, including lower expression of inhibitory receptors and higher expression of costimulatory molecules. Notably, the TCF1 + subset displays exclusive proliferative potential and could differentiate into TCF1 - effector cells upon antigenic stimulation. Pathway analyses support the role of TCF1 + and TCF1 - subsets as resource cells and effector cells, respectively. Furthermore, the TCF1 + CD8 + T cell subset is primarily present in the spleen and exhibits a resident phenotype. These findings provide insight into the differentiation of allogeneic and xenogeneic CD8 + T cells and have implications for the development of immunotherapeutic strategies targeting acute GvHD.