In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3 + regulatory T cells (T regs ) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate T reg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3 ζ /T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect T regs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and T regs in models of multiple myeloma (MM)-where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated T reg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on T reg homeostasis within an alloimmune context. Specifically, we studied the induction of T regs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3 + T regs . Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3 + T reg responses and, by extension, stipulate an additional means of facilitating T reg fitness via type I IFNs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Eskandari, Allos, Safadi, Sulkaj, Sanders, Cravedi, Ghobrial, Berger and Azzi.)