Abstract As invaluable as the standard 2-dimensional (2D) monolayer in vitro cell culture system has been, there is increasing evidence that 3-dimensional (3D) non-adherent conditions are more relevant to the in vivo condition. While one of the criteria for human mesenchymal stem cells (MSCs) has been in vitro plastic adherence, such 2D culture conditions are not representative of in vivo cell-cell and cell-extracellular matrix (ECM) interactions, which may be especially important for this progenitor/stem cell of skeletal and connective tissues. The 3D spheroid, a multicellular aggregate formed under non-adherent 3D in vitro conditions, may be particularly suited as an in vitro method to better understand MSC physiological processes, since expression of ECM and other adhesion proteins are upregulated in such a cell culture system. First used in embryonic stem cell in vitro culture to recapitulate in vivo developmental processes, 3D spheroid culture has grown in popularity as an in vitro method to mimic the 3-dimensionality of the native niche for MSCs within tissues/organs. In this review, we discuss the relevance of the 3D spheroid culture for understanding MSC biology, summarize the biological outcomes reported in the literature based on such this culture condition, as well as contemplate limitations and future considerations in this rapidly evolving and exciting area.