FOXP3⁺ regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4⁺CD25⁻EGFP⁻ T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4⁺FOXP3⁻ T cells into functional CD4⁺FOXP3⁺ Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.