In this report, we introduce a standard protocol for stem cell self-renewal in vitro. Both fundamental and major procedures of stem cell manipulation, which are required for somatic cell coculture and self-renewal, are briefly described since they are important for stabilization and data normalization. In this chapter, information on the basic preparation of stem cell culture such as labware washing, equipment sanitization, microbe control, and mycoplasmosis prevention is provided. In addition, protocols for cell retrieval and preservation, proliferation assays, and basic manipulation techniques for the coculture of stem cells with somatic cells are described.