Generation of regulatory T cells (or Treg) derived hybridomas offers a tool to study their antigen specificity. T cells hybridomas are produced by fusing TCR α-β-thymoma BW5147 with highly dividing T cell population. In vitro anergy of Tregs is an obstacle in generation of highly dividing Treg population for their fusion. In this chapter, we describe a simple and efficient method to generate large number of blasting Treg and their successful fusion with thymoma BW5147. The resultant hybridomas lose Treg-specific transcription factor FoxP3, respond to antigenic stimulation by producing IL-2, and thus allow the evaluation of antigen specific, Tregs-derived TCRs.