Abstract Stem cells have emerged as important players in the generation and maintenance of many tissues. However, the accurate in vitro simulation of the native stem cell niche remains difficult due at least in part to the lack of a comprehensive definition of the critical factors of the stem cell niche based on in vivo models. Three-dimensional (3D) cell culture systems have allowed the development of useful models for investigating stem cell physiology particularly with respect to their ability to sense and generate mechanical force in response to their surrounding environment. We review the use of 3D culture systems for stem cell culture and discuss the relationship between stem cells and 3D growth matrices including the roles of the extracellular matrix, scaffolds, soluble factors, cell-cell interactions and shear stress effects within this environment. We also discuss the potential for novel methods that mimic the native stem cell niche in vitro as well as the current associated challenges.