Clinical benefits of umbilical cord blood (UCB) grafts include the lower incidence of acute GVHD following allogeneic transplantation. We have previously demonstrated dramatically lower expression of the NFAT1 transcription factor and its associated transcripts and known binding partners in UCB CD4+ cells following primary stimulation when compared to T-cells obtained from adult blood (AB). We hypothesize that NFAT1 and NFAT1-dependent factors play a critical role in the increased proliferation and decreased cytokine production seen in UCB T-cells. We have now demonstrated in vitro via RNAi-mediated knockdown in AB-derived primary T-cells with RT-PCR and Cytometric Bead Assay that expression of a GVHD-promoting, Th1-type cytokine profile is dependent on expression of NFAT1, and that knockdown of NFAT1 results in a transcriptional and cytokine profile similar to that seen in UCB. However, microarray and RT-PCR time point analyses indicate NFAT1 transcripts are not changed in UCB versus AB CD4+ cells. This may suggest a crucial regulatory role for post-translational events which may preferentially occur in UCB-derived T-cells in vivo. Indeed, we have observed ubiquitination of NFAT1 and rapid rescue of NFAT1 expression following treatment with proteasome inhibitor in UCB-derived T-cells in vitro. NFAT1 in UCB CD4+ cells is shown by co-IP to be significantly more ubiquitinated than in AB-derived cells. Overall, our data indicates that regulation of NFAT1 through ubiquitination and proteasomal degradation, and its subsequent downstream effects may constitute a key difference between T-cells of adult and neonatal origin; specifically that this post-translational modification may serve as a method by which UCB-derived T-cells may prevent NFAT1 activity, impacting their allogeneic response.