First, we compared different protocols involving simultaneous and non-overlapping schedules by infusing IL-2 either free or complexed with anti-JES6mAb (IAC) and a TL1A-Ig fusion protein (FP) into normal B6-FoxP3RFP mice. A highly reproducible protocol was developed, wherein FP was administered first, followed by IAC. This routinely resulted in ~50% FoxP3+/CD4+ T cells, and also an overall increase in the CD4/CD8 ratio in these animals versus controls (3.7 vs. 1.5 in lymph node and 4.1 vs. 1.7 in splenic T cells). Notably, expanded Tregs exhibited elevated CD25 expression and there was a 4-5x increase in the KLRG1+ fraction of FoxP3+CD4+ T cells. The expanded Treg population in these mice diminished by 50% in the first 4 days following cessation of treatment and returned to normal levels within 2 weeks. We then examined transplant outcome after obtaining cells from these Treg expanded mice as donors for an allogeneic, complete MHC-mismatched HSCT. Lymph node cells from Treg-expanded B6-FoxP3RFP (H2b) mice were transplanted together with normal TCD-BM into lethally conditioned BALB/c (H2d) recipients. Recipients were treated with different regimens of TL1A-Ig and/or free IL-2 (10,000 units/mouse) early post-HSCT. Mice were monitored for weight change, clinical score and survival. Treg frequencies were determined in the blood and tissues 1 week post-HSCT. Initial TL1A-Ig treatment of recipients following transplant with Treg expanded donor T cells resulted in rapid lethality. These observations were consistent with the known effector cell co-stimulation via TNFRSF25 in the presence of antigen. Only Treg expansion in the donor combined with initial IL-2 followed by TL1A-Ig treatment of the recipient post-HSCT resulted in amelioration of GVHD. In summary, a combination strategy employing the targeted signaling of CD25 and TNFRSF25 was identified which can markedly expand Treg cells including a KLRG1+ population. Signaling via TNFRSF25 followed by CD25 in T cell donors in conjunction with the reverse targeting of these receptors in recipients post-transplant resulted in attenuation of GVHD.