Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. The role of Th17 cells in its pathophysiology remains a matter of debate. In this study, we assessed whether enrichment of human peripheral blood mononuclear cells (PBMCs) with in vitro Th17-polarized CD4⁺ T cells would exacerbate xenogeneic GVHD (xGVHD) into NOD-scid IL-2Rγ null (NSG) mice. Naive human CD4⁺ T cells were stimulated under Th17-skewing conditions for 8 to 10 days and then coinjected in NSG mice with fresh PBMCs from the same donor. We observed that Th17-polarized cells engrafted and migrated toward xGVHD target organs. They also acquired a double-expressing IL-17A⁺IFNγ⁺ profile in vivo. Importantly, cotransfer of Th17-polarized cells (1 × 10⁶) with PBMCs (1 × 10⁶) exacerbated xGVHD compared with transplantation of PBMCs alone (2 × 10⁶). Furthermore, PBMC cotransfer with Th17-polarized cells was more potent for xGVHD induction than cotransfer with naive CD4⁺ T cells stimulated in nonpolarizing conditions (Th0 cells, 1 × 10⁶ + 1 × 10⁶ PBMCs) or with Th1-polarized cells (1 × 10⁶ + 1 × 10⁶ PBMCs). In summary, our results suggest that human Th17-polarized cells can cooperate with PBMCs and be pathogenic in the NSG xGVHD model.