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Summary: Lymphoid tissue stromal cells are important regulators of spleen homeostasis and immune responses. Here, we present an optimized protocol that describes the digestion and enrichment steps for the isolation and analysis of rare populations of stromal cells, including fibroblastic reticular cells, perivascular cells, and glial cells found in the spleen. This protocol is suitable for subsequent analysis of spleen stromal cells by flow cytometry or single-cell RNA sequencing and to analyze different disease models.For complete details on the use and execution of this protocol, please refer to Alexandre et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.