Given the potential importance of human pluripotent stem cells (hPSCs) in translational research and regenerative medicine, the aim of the present study was to develop a simple, safe, and cost-effective substrate to expand hPSCs. We report the development of an extracellular matrix (ECM), designated "RoGel," based on conditioned medium (CM) of human fibroblasts under serum- and xeno-free culture conditions. The long-term self-renewal of hPSCs on RoGel was also assessed. The results showed that self-renewal, pluripotency, plating efficiency, and cloning efficiency of hPSCs on this newly developed ECM were similar to those of Matrigel, the conventional mouse-cell line-derived ECM. The cells had the capability to passage mechanically on a cold surface, which resulted in their long-term maintenance with normal karyotype. We have demonstrated that CM-coated plates preserved for 1 year at room temperature maintained the capability of hPSC expansion. This ECM provides an attractive hPSC culture platform for both research and future therapeutic applications.