The adoptive transfer of CD4(+)CD25(+) natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high-grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell-specific surface markers. Depletion of CD127(+) cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3(+) cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4(+)CD25(+)CD127(low) Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA(-)FOXP3(+) memory-type Treg as the main source of converting cells, whereas CD45RA(+)FOXP3(+) Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell-type-specific characteristics after repetitive TCR stimulation.