We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel(™) and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH(3), OH, COOH and NH(2). hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.