In recent years there has been a growing interest in culturing adherent cells using three-dimensional (3D) techniques, rather than more conventional 2D culture methods. This interest emerges from the realization that growing cells on plastic surfaces cannot truly re-create 3D in vivo conditions and therefore might be limiting the cells' potential. In addition, adult stem cells exist in specialized microenvironments, or niches, where the spatial organization of different niche elements (such as different cell types, extracellular matrix) contributes significantly to stem cell maintenance, which cannot be represented using 2D in vitro models. We have generated a range of different 3D approaches for the analysis of mesenchymal stem cells (MSCs) using both mono- and co-culture environments.