Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7-8, with return to near baseline after 2-3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.