Preconditioning with the nonmyeloablative regimen total lymphoid irradiation (TLI) before hematopoietic cell transplantation facilitates the establishment of mixed chimerism and protects against graft-versus-host disease. We reported that the development of mixed chimerism requires interleukin (IL)-4 and is associated with increased host anti-donor TH2 cells, but the effect of TLI on the differentiation of immunocompetent donor cells has not been investigated. To examine the extent to which TLI preconditioning influences donor T cells, we measured responses of transgenic CD4+cells specific for ovalbumin peptide (OVA-Tg) following in vivo and in vitro antigen stimulation in a TLI-preconditioned environment. OVA-Tg cells that were adoptively transferred into TLI-preconditioned mice that express cross-reactive antigens produced more IL-4 and less interferon-gamma and IL-2 than controls when stimulated with OVA peptide one week later. OVA-Tg primed in vitro with spleen from TLI-preconditioned mice generated more TH2 and fewer TH1 cells when stimulated in recall enzyme-linked immunosorbent spot (ELISPOT) assays with OVA peptide. Naive OVA-Tg up-regulated CD69 and CD25 normally following stimulation with OVA peptide in the presence of spleen from TLI-preconditioned mice, but proliferated less and secreted less IL-2 than controls. Surprisingly, naive OVA-Tg secreted IL-4 in primary cultures that were stimulated with OVA peptide in the presence of spleen from TLI-preconditioned mice. This response depends on CD4+cells from TLI-spleen, which constitutively produce IL-4 and are composed primarily of CD4+-natural killer T (TNK) cells. Thus, TLI preconditioning enriches for IL-4-secreting and TNK-like CD4+cells that may function in the protection from graft-versus-host disease by redirecting the differentiation of immunocompetent donor CD4+cells toward TH2 and away from pathogenic TH1 cells.