Mesenchymal stem cells (MSCs) are a promising cell source for tissue repair and regeneration due to their multilineage capacity, potential for autologous use, and secretion of potent bioactive factors to catalyze the endogenous repair program. However, a major limitation to current cell-based tissue engineering approaches is the drastic loss of cells upon transplantation. The causation of this loss, whether due to apoptosis following a dramatic change in the microenvironment or migration away from the defect site, has yet to be determined. MSCs formed into aggregates, known as spheroids, possess a strong therapeutic advantage compared to the more commonly used dissociated cells due to their improved resistance to apoptosis and increased secretion of endogenous trophic factors. Furthermore, the use of biomaterials such as alginate hydrogels to transplant cells in situ improves cell survival, localizes payloads at the defect site, and facilitates continued instruction of cells by manipulating the biophysical properties of the biomaterial. Transplantation of MSC spheroids without a vehicle into tissue defects comprises the majority of studies to date, ceding control of spheroid function due to the cell’s interaction with the native tissue extracellular matrix and abrogating the established benefits of spheroid formation. Thus, there is a significant need to consider the role of biomaterials in transplanting MSC spheroids using an appropriate carrier. In this chapter, we describe high-throughput formation of spheroids, steps for further characterization, and encapsulation in alginate hydrogels with an eye toward localizing MSC spheroids at the target site.