Background <p>Dental stem cells hold significant potential in regenerative medicine due to their multipotency, accessibility, and immunomodulatory effects. Flow cytometry is a critical tool for analyzing these cells, particularly in identifying and characterizing immunomodulatory markers that enhance their clinical applications. This systematic review aims to answer the question:&#xa0;<i>"How does flow cytometry facilitate the identification and characterization of immunomodulatory markers in dental stem cells to enhance their application in regenerative medicine?"</i>.</p> Methods <p>An exhaustive literature search was conducted in PubMed, retrieving 430 studies, of which 284 met inclusion criteria. Studies were selected based on the use of flow cytometry to analyze immunomodulatory markers in dental stem cells, focusing on methodologies, key findings, and challenges.</p> Results <p>Of the 284 articles, 229 employed flow cytometry, with 115 reporting relevant results. Flow cytometry revealed important insights into the immunological interactions of various dental stem cells, including dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, and stem cells from the apical papilla, by identifying and characterizing immunomodulatory markers such as PD-L1, IDO, and TGF-β1.</p> Conclusions <p>Flow cytometry is essential for advancing the understanding of dental stem cells' immunomodulatory properties. Standardization of methodologies is required to overcome technical challenges and enhance the clinical applications of dental stem cells in regenerative medicine and immunotherapy.</p> Graphical Abstract <p></p>