The majority of regulatory T cells (T(regs)) are believed to be of thymic origin. It has been hypothesized that this may result from unique intrathymic environmental cues, possibly requiring a dedicated antigen-presenting cell (APC). However, T cell-intrinsic developmental regulation of the susceptibility to T(reg) differentiation remains a mutually non-exclusive scenario. We found that upon exposure of monoclonal T cells of sequential developmental stages to a thymic microenvironment expressing cognate antigen, the efficiency of T(reg) induction inversely correlated with progressive maturation. This inclination of immature thymocytes toward T(reg) differentiation was even seen in an APC-free in vitro system, providing only TCR stimulation and IL-2. In support of quantitative but not qualitative features of external cues being critical, thymic epithelial cells as well as different thymic dendritic cell (DC)-subtypes efficiently induced T(reg) development of immature thymocytes, albeit at strikingly different optimal doses of cognate antigen. We propose that the intrinsically high predisposition of immature thymocytes to T(reg) development may contribute to the predominantly thymic origin of the T(reg) repertoire. The underlying instructive stimulus, however, does not require unique features of a dedicated APC and can be delivered by hematopoietic as well as epithelial thymic stromal cells.