Abstract Thymic epithelial cells (TECs) are required for the development and differentiation of T cells and are sufficient for the positive and negative selection of developing T cells. Although TECs play a critical role in T cell biology, simple, efficient and readily scalable methods for the transfection of TEC lines and primary TECs have not been described. We tested the efficiency of Nucleofection for the transfection of 4 different mouse thymic epithelial cell lines that had been derived from cortical or medullary epithelium. We also tested primary mouse thymic epithelial cells isolated from fetal and postnatal stages. We found that Nucleofection was highly efficient for the transfection of thymic epithelial cells, with transfection efficiencies of 30-70% for the cell lines and 15-35% for primary TECs with low amounts of cell death. Efficient transfection by Nucleofection can be performed with established cortical and medullary thymic epithelial cell lines as well as primary TECs isolated from E15.5 day fetal thymus or postnatal day 3 or 30 thymus tissue. The high efficiency of Nucleofection for TEC transfection will enable the use of TEC lines in high throughput transfection studies and simplifies the transfection of primary TECs for in vitro or in vivo analysis.