Human thymic stromal lymphopoietin (TSLP) activates dendritic cells (DCs), which promote the proliferation and differentiation of CD4+ T cells. However, murine TSLP (mTSLP) can act directly on CD4+ T cells and bring about their differentiation. We studied the role of mTSLP in the generation of CD4+CD25+FoxP3+ T cells from thymocytes. mTSLP promoted the differentiation of CD4+ single-positive thymocytes into CD4+CD25+FoxP3+ T cells. Although we cannot exclude an effect of TSLP mediated through DCs due to co-stimulatory effects, mTSLP appears to act directly on thymocytes. T-cell receptor and TSLP receptor signaling act synergistically on thymocytes to generate CD4+CD25+FoxP3+ T cells. mTSLP may play an important role in maintaining immune tolerance by promoting the conversion of thymocytes into natural regulatory T cells via escape from negative selection.