As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3), or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.