Abstract Cdk2 has a central role in cell cycle re-entry and Cdk2 inhibition is mandatory for induction of T cell tolerance. To determine the effect of Cdk2 inhibition on T cell alloresponses we used the B6D2F1 model of bone marrow transplantation (BMT) and two Cdk2 inhibitors (Cdk2i), CYC202 and CYC205. B6D2F1(Kd) recipients infused with BM and T cells from C57BL/6(Kb) donors were treated with each Cdk2i for 3 weeks and graft versus host disease (GvHD) was assessed over a 70-day period. Cdk2i treatment decreased GvHD mortality (p=0.0054). To examine if Cdk2i affected Treg differentiation we used T cells from Foxp3.GFP-KI mice during BMT. Cdk2i-treated recipients had no difference in CD4+GFP+ Treg compared to control groups in peripheral blood, splenocytes, lymph nodes and intestinal lymphoid cells (ILC) but had increased CD8+GFP+ Treg, predominantly in ILCs (p=0.05). In Treg polarizing culture Cdk2i had only minimal effect on CD4+GFP+ cells but induced significant increase of CD8+GFP+ cells. However, TGF-b-mediated signaling, as determined by Smad3 phosphorylation was unaffected. FOX family members are regulated by transcriptional and epigenetic mechanisms. The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2) induces histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. Cdk2 phosphorylates EZH2 Thr350, which is important for maintenance of H3K27me3 at EZH2 target loci. We determined that Cdk2i inhibited EZH2 Thr350 phosphorylation in CD8+ T cells, which coincided with increased Foxp3 expression. ChIP-PCR identified EZH2 binding on three sites of Foxp3 promoter, which was abrogated by Cdk2i. Thus, Cdk2 inhibition induces CD8+ Treg via EZH2-mediated epigenetic mechanisms.