Abstract: Tertiary lymphoid structures (TLS) are organized ectopic lymphoid tissues formed by lymphocyte aggregation in pathological environments. Mature TLS, including B-cell regions surrounded by T cells and germinal centers, have been identified in a wide variety of tumors. TLS offer sites of interaction between cancer cells and immune system cells in the tumor microenvironment (TME), and are closely associated with patient prognosis. It is thus necessary to induce TLS formation to allow T and B cells to develop and differentiate into effector and memory cells that recognize and kill cancer cells, and mediate anti-tumor immune responses. This review focuses on TLS formation and composition, and summarizes and compares existing techniques. We also emphasize the potential predictive roles of TLS development, location, and internal components as biomarkers of response to immunotherapy, and potential therapeutic strategies that induce TLS formation.