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.