T cells go through most of their maturation in the thymus, and the stromal constituents of the thymus are therefore essential for T cell differentiation. The thymic stroma secretes the factors that recruit and sustain T cell progenitors, and they also partake in the shaping of a functional and tolerant T cell receptor repertoire. The damage incurred to the thymic stromal compartment by bone marrow conditioning regimens as well as by the natural aging process impairs T cell production. Yet little is known of how to prevent or reverse this damage. The development of high-throughput, single-cell analysis technologies has enabled better characterization of thymic stromal cells. This does however require tissue dissociation protocols optimized for stromal cell isolation. In this chapter, we detail the methodology of harvesting thymus stromal cells from human and murine tissue for downstream applications such as flow cytometric analysis and single-cell RNA sequencing. © 2023. Springer Science+Business Media, LLC, part of Springer Nature.