In hematopoietic stem cell transplantation (HSCT), donor-derived allogeneic T cells contribute to the graft-versus-tumor (GVT) effect but can also trigger graft-versus-host disease (GvHD), a major source of post-transplant morbidity and mortality. Regulatory T cells (Tregs), particularly FoxP3⁺ Tregs, play a pivotal role in promoting immune tolerance and preventing GvHD, as established in murine models. Translating these findings to human therapy requires the isolation and expansion of pure, stable Treg populations suitable for clinical infusion. This study investigates whether clinical-grade CD45RA⁺ naïve Tregs—known for their stem-like properties and enhanced persistence—can be isolated and expanded while retaining their phenotype and immunosuppressive function. Peripheral blood mononuclear cells (PBMCs) were used to isolate naïve Tregs defined as CD45RA⁺CD4⁺CD25⁺CD127⁻. Artificial antigen-presenting cells (aAPCs) derived from genetically engineered K562 cells expressing CD64, CD86, and CD137L were loaded with OKT3 monoclonal antibody to stimulate Treg cell expansion. Tregs were re-stimulated on days 0 and 10 and assessed on day 21 using flow cytometry, CFSE suppression assays, and TCR repertoire analysis. Results showed that CD45RA⁺ Tregs expanded approximately 500-fold, compared to 200-fold for CD45RA⁻ Tregs, with >80% maintaining FoxP3 expression. Cytokine production remained low, with <5% IL-2 and <2% IFNγ and TNFα. TCR analysis revealed a maintained broad polyclonal repertoire, supporting the diversity and functional stability of expanded Tregs. This study demonstrates a feasible and scalable method for generating clinical-grade, stable, and suppressive CD45RA⁺ Tregs suitable for immunotherapy. These findings support further clinical trials evaluating their efficacy in preventing or treating GvHD in HSCT recipients.