The expression of the integrin αE (CD103), may enhance the retention of regulatory T cells to peripheral inflammatory sites and possibly contribute to their suppressive potential. The aim of this study was to define the regulatory role of IL-2 and TGF-β1 on the CD103 expression and the optimal in vitro conditions for the induction/expansion of human CD4+ and CD8+ Tregs. Cord blood mononuclear cells (CBMC) were stimulated under various culture conditions, including anti-CD3, anti-CD28, IL-2 and TGF-β1. TGF-β1 and IL-2 were both required for optimal expression of CD103. In addition, TGF-β1 and IL-2 synergistically induced CD103 expression on CD8+ T cells, whereas, only additive induced expression was noted on CD4+ T cells. Surprisingly, CD103 expression was not dependent upon CD28 costimulation. IL-2 also played a central role in CD103 expression by CD25hi Foxp3+ Tregs. IL-2, TGF-β1 and anti-CD3 defined the optimal stimulatory conditions favouring the induction/expansion of both CD4+ and CD8+ human Tregs from naive CBMC. Thus, this study provides new insights into the regulatory role of IL-2 upon CD103 expression by human cord blood CD4+ and CD8+ T cells. Furthermore, it identifies the in vitro culture conditions driving the differentiation of the novel phenotype CD4+ and CD8+ CD103+ CD25hi Foxp3+ Tregs from human CBMC.