T-cell-depleting strategies are an integral part of immunosuppressive regimens used in the hematological and solid organ transplant setting. Besides prevention of alloreactivity, treatment with rabbit antithymocyte globulin (rATG) has been related to the induction of immunoregulatory T cells (Treg) in vitro and in vivo. To investigate Treg induced by rATG, we prospectively studied the effect of rATG induction therapy in liver-transplanted recipients in vivo (n = 28). Treg induction was further evaluated by means of Treg-specific demethylation region (TSDR) analysis within the FOXP3 locus. Whereas no induction of CD4+ CD25highCD127− Treg could be observed by phenotypic analysis, we could demonstrate an induction of TSDR+ T cells within CD4+ T cells exclusively for rATG-treated patients in the long-term (day 540) compared with controls (P = NS). Moreover, although in vitro experiments confirm that rATG primarily led to a conversion of CD4+ CD25− into CD4+ CD25+ T cells displaying immunosuppressive capacities, these cells cannot be classified as bona fide Treg based on their FOXP3 demethylation pattern. Consequently, the generation of Treg after rATG co-incubation in vitro does not reflect the mechanisms of Treg induction in vivo and therefore the potential clinical relevance of these cells for transplant outcome remains to be determined.