Targeting costimulatory interactions between T cells and APC can potenti ally be useful to treat auto-immune diseases and to prevent transplant r ejection. Blocking of these interactions has already been shown to lead to tolerance in several autoimmunity and transplantation models, but the underlying mechanisms are incompletely understood. Here, we targeted tw o particular costimulatory pathways (the CD28 CD80/CD86 and the CD40 - CD40L pathway) and tried to elucidate the mechanisms responsible for pr omoting tolerance when both pathways are blocked. More specifically, we investigated the involvement of regulatory T cells and of deletion of al lo-reactive cells in the induction and maintenance of tolerance after co stimulation blockade. To study the mechanisms responsible for promoting tolerance after costim ulation blockade, a MHC-mismatched parent-in-F1 mouse model of GVHD was developed. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F1 offspring (C57BL/6xC3H) induced a GVHD chara cterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4 -Ig every three days during three weeks after splenocyte injection preve nted disease development and induced a long lasting state of stable mixe d chimerism (> 120 days). In parallel, host-specific tolerance was achie ved as demonstrated by lack of host-directed allo-reactivity of donor-ty pe T cells both in vitro and in vivo. Chimerism and tolerance were also obtained after transfer of CD25+ cell depleted splenocytes, showing that CD25+ natural Treg cells are not essential for tolerance induction. We further showed that costimulation blockade results in enhanced Treg cell activity at early timepoints (day 6 day 30) after splenocyte transfer . This was demonstrated by the presence of a high percentage of Foxp3+ c ells among donor CD4+ cells in the spleen of treated animals, and the fi nding that isolated donor T cells at an early timepoint (day 30) after s plenocyte transfer displayed suppressive capacity in vitro. When sorted Foxp3- splenocytes were used as donor cells, there was no de novo induct ion of Foxp3+ cells, indicating that the high percentage of Foxp3+ cells among donor cells originated from Foxp3+ cells present in the donor cel l inoculum. At later timepoints (> 30 days after splenocyte transfer), d eletion of host-reactive T cells was found to be a major mechanism respo nsible for tolerance. Finally, we provided evidence that IL-10 is involv ed in the induction of tolerance by costimulation blockade. In the absen ce of IL-10 producing donor cells, costimulation blockade could not prev ent lethal GVHD in most of the recipient animals.