The development of a three-dimensional (3D) culture system is very important for regenerative medicine and drug discovery applications of stem cell technology because the 3D culture condition could mimic the stem cell environment in vivo and support accurate differentiation. This chapter describes a novel 3D culture technique for efficient induction of adipogenic differentiation. This 3D culture system provides an easy way to allow cells to form a 3D spheroid structure without any matrix derived from animal and chemical substances. We firstly describe the details of the 3D culture technique using human mesenchymal stem/progenitor cells (MPCs) and its optimization. Then we elaborate on the protocol of efficient induction for adipogenic differentiation and adipocyte-specific gene expression, including peroxisome proliferator-activated receptor-γ, with our culture system in human MPCs.