Human mesenchymal stem cells, such as human adipose-derived stem cells (hASCs), are typically cultured on a two-dimensional (2D) monolayer material surface, on which 2D culturing methods are easily performed and time-saving. However, hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2D monolayer culturing method compared to hASCs cultured with a three-dimensional (3D) culturing method, such as suspension culture. In this study, we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2D and 3D culturing processes. The hASCs cultivated with a 3D culturing process after 2D culture showed at least 2-fold enhanced pluripotency (Sox2, Nanog, and OCT4) compared with that of hASCs cultured with the 2D culture process alone. Furthermore, hASCs obtained from the 3D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2D culture process when hASCs were induced to differentiate. However, their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3D culture to 2D culture and vice versa, which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2D and 3D culture. The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2D and 3D culturing processes. Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs, which may reduce the effects of the aging process in hASCs. We discuss whether the environment of stem cell culture (i.e., 2D or 3D cultivation) can affect stem cell fate in terms of pluripotency and differentiation reversibility.