T regulatory cells (Tregs) suppress immune responses and therefore have potential to be used in the clinic as a cellular therapy for autoimmune disease and to prevent rejection of transplanted organs. Obtaining sufficient numbers of these cells for therapeutic use is a challenge, however, since there are currently no Treg cell-specific markers, and they have a poor in vitro expansion potential. Tregs express high levels of FOXP3, a transcription factor that is critical for their function. We have shown that lentivirus-based overexpression of FOXP3 can reprogram na�ve or memory human CD4+ T cells into cells which possess a phenotype and function similar to ex vivo Tregs. Here we will review the methodology involved in generating, expanding, and testing FOXP3-transduced cells and their ex vivo Treg counterparts.