Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX + cells has not been developed. Here we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato + cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato + cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD , and COL1A1 . Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.