AbstractAdoptive regulatory T‐cell (Treg) transfer has emerged as a promising therapeutic strategy for regulating immune responses in organ transplantation, graft versus host disease, and autoimmunity, including Type 1 diabetes. Traditionally, Treg for adoptive therapy have been sorted and expanded in vitro using high doses of IL‐2, demonstrating stability and suppressive capabilities. However, limitations in their long‐term survival post‐infusion into patients have been observed. To address this challenge, we investigated a novel expansion protocol incorporating interleukin‐7 (IL‐7) alongside the traditional method utilizing IL‐2 (referred to as IL‐7 method, IL‐7M). Our study revealed that naïve Treg express significant levels of CD127 and display robust responsiveness to IL‐7, characterized by STAT‐5 phosphorylation. Expanding naïve Treg with the IL‐7M protocol led to a substantial enrichment of CD45RA⁺CD62L⁺CD95⁺ Treg but showing a reduction in the final cell yield and suppressive function. Moreover, Treg expanded with the IL‐7M exhibited preserved telomere length and demonstrated enhanced resistance to cytokine withdrawal and fas‐mediated apoptosis. When transferred into NSG mice IL‐7M‐Treg persisted longer and reduced the expansion of T cells, but did not significantly reduce the severity of xenoGvHD. In conclusion, our data demonstrate the feasibility of expanding naïve Treg in the presence of IL‐7 to generate a Treg product enriched in poorly differentiated CD45RA⁺ cells with enhanced survival capabilities.