Mesenchymal stem cells (MSCs) can give rise to many tissue types including bone, cartilage, muscle, and fat. For that reason, MSCs have found many research applications in tissue engineering and regenerative medicine. In recent years, rapid development of novel in vitro and ex vivo approaches have contributed to our understanding of the molecular mechanisms of MSC lineage specification. In particular, it is now widely agreed that the standard practice of in vitro cell differentiation using monolayer 2D cultures does not provide sufficient microenvironmental cues to ascertain proper self-renewal and differentiation of MSCs into various cell lineages. This collection highlights experimental protocols that incorporate mechanical and other molecular cues which model the microenvironment of the tissues of interest. These protocols enhance the efficiency and fidelity of MSC differentiation and tissue engineering. The goal of the collection is to lower the threshold for researchers interested in utilizing these approaches.