Background: Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8⁺ T (T_RM) cells play a key role. The mechanisms involved in the activation of CD8⁺ T_RM cells during allergic flare-up responses are not understood. Methods: The expression of CD100 and its ligand Plexin B2 on CD8⁺ T_RM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice. Results: We show that CD8⁺ T_RM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1β and decreased recruitment of neutrophils to the epidermis. Conclusion: Our study demonstrates that CD100 is expressed on CD8⁺ T_RM cells and is required for full activation of CD8⁺ T_RM cells and the flare-up response of ACD.