Human mesenchymal stromal cells (hMSCs) play a crucial role in tissue engineering and regenerative medicine and thus have important clinical potential for cell-based therapy. However, the limited cell number and the difficulty in detecting these cells in vivo have restricted many hMSC studies. Therefore, the development of hMSCs immortalized with telomerase and expressing red fluorescence protein will facilitate their expansion and detection in vivo, and these cells will be important for both to stem cell research and clinical use. In this chapter, we describe the protocols used to establish telomerase- and red fluorescence protein-expressing immortalized hMSCs using a nonviral transfection method. These cells will be useful tools for stem cell research and translational studies.