Reprogramming T cells to adopt regulatory T cell (Treg) functions represents a promising strategy for treating autoimmune disorders and graft-versus-host disease. The development and maintenance of Treg cells is dependent on FOXP3 expression, which in turn is tightly controlled through epigenetic modification of intronic conserved non-coding sequences (CNS) surrounding the first coding exon. Two major challenges associated with FOXP3 gene-transfer approaches for Treg reprogramming are: achieving adequately high FOXP3 expression, and avoiding eventual gene silencing. We devised two gene editing strategies with the goal of driving stable expression of FOXP3 in primary human T cells. Using a combination of TALEN-mediated gene disruption and adeno-associated-virus (AAV) delivered donor repair templates, we introduced either an MND promoter upstream of the first coding exon, or we deleted intronic CNS implicated in transcriptional silencing. The donor templates were also designed to co-express FOXP3 with either GFP or EGFRt (truncated Epidermal Growth Factor receptor), to allow tracking and purification of edited cells. Both strategies resulted in T cells stably expressing FOXP3 at high levels (~60 %). Introduction of the MND promoter resulted in the greatest levels of cellular FOXP3 expression (MFI), and these cells showed phenotypic and functional changes consistent with Treg cells, including: surface marker expression (CD25high, CD127low, CTLA4high, LAG3high), suppression of cytokine production (IL-2, IL-17 and IFN-γ), and resistance to Rapamycin. Edited cells also suppressed the proliferation of stimulated T cells in vitro, demonstrating their effective ‘reprogramming’ towards a Treg lineage. Thus, our gene modification strategy allowed us to over-ride (using the MND promoter) or modulate (by deleting CNS elements) endogenous FOXP3 regulatory mechanisms to enforce stable, long-term FOXP3 expression in T cells that were not previously committed to the Treg lineage. This approach, used alone or in combination with selection for disease-relevant TCR specificity or with delivery of a chimeric antigen receptor, is likely to be broadly applicable for producing stable, functionally active Tregs for a range of future clinical applications.