Donor treatment with granulocyte-colony-stimulating factor (G-CSF) attenuates the ability of donor T cells to induce acute graft-versus-host disease (aGVHD) but increases the severity of chronic GVHD (cGVHD). We investigated the role of the regulatory cytokine transforming growth factor β (TGF-β) in this paradox in well-established murine models of aGVHD and cGVHD wherein recipients undergo transplantation with splenocytes from donors treated with G-CSF. Neutralization of TGF-β after stem-cell transplantation (SCT) significantly increased the severity of aGVHD, and the concurrent prevention of interleukin-10 (IL-10) production further exaggerated this effect. Early after SCT, donor T cells were the predominant source of TGF-β and were able to attenuate aGVHD in a TGF-β-dependent fashion. Although the neutralization of TGF-β augmented the proliferation and expansion of donor T cells after SCT, it paradoxically impaired cellular cytotoxicity to host antigens and associated graft-versus-leukemia (GVL) effects. In cGVHD, neutralization of TGF-β from day 14 after SCT attenuated histologic abnormalities, and CD11b⁺ mononuclear cells infiltrating sclerodermatous skin produced 50-fold more TGF-β than corresponding T cells. Thus, though the production of TGF-β by donor T cells early after transplantation attenuates aGVHD and is required for optimal GVL, the production of TGF-β late after SCT is preferentially from mononuclear cells and mediates cGVHD. These data have important implications for the timing of therapeutic TGF-β neutralization to prevent cGVHD after allogeneic SCT. (Blood. 2005;106:2206-2214)