Abstract Atopic dermatitis (AD) is a debilitating type 2 inflammatory disease of the skin characterized by pruritus. Mast cells (MCs) and eosinophils (eos) are elevated in AD tissue. Siglecs are inhibitory receptors found on immune cells, with Siglec-8 selectively expressed on MCs and eos. Engagement of Siglec-8 with antibody results in broad inhibition of MC activation (IgE-dependent and -independent pathways). MCs and eos are key drivers of itch. Crosstalk between skin MCs and sensory neurons contributes to non-histaminergic itch via activation of Mas-related G-protein receptor X2 (MRGPRX2). MRGPRX2-mediated MC activation has been implicated in chronic urticaria and allergic contact dermatitis, however, this mechanism has not been studied in the pathogenesis of AD. We hypothesize that activation of MCs through MRGPRX2 contributes to chronic inflammation and itch in patients with AD. Supernatants were collected from intact ex vivo cultured lesional and non-lesional skin biopsies from donor-matched patients with AD or healthy volunteers. Following ex vivo culture, secreted mediators were quantified using ELISA and meso-scale discovery (MSD). Biopsies were enzymatically digested and MRGPRX2 expression and function were measured using flow cytometry and MSD. Acute MRGPRX2 activation in vivo and the effects of a Siglec-8 monoclonal antibody (mAb), compared with isotype control, were studied in Siglec-8 transgenic mice. MC and eosinophil numbers were increased in lesional biopsies from patients with AD compared with patient-matched non-lesional and healthy volunteer control biopsies. MCs from AD lesional biopsies showed significant evidence of activation and degranulation compared to MCs in non-lesional biopsies. Levels of MC-specific proteases, inflammatory and pruritic mediators were significantly elevated in supernatants from ex vivo cultured lesional biopsies, including CPA3, CCL17 (TARC), IL-8, IL-13 and IL-31. Levels of MRPGRX2 ligands in supernatants from AD lesional biopsies were significantly elevated. Ex vivo challenge of healthy skin tissue with MRGPRX2 ligands partially replicated the AD lesional microenvironment. Lastly, intradermal injection of MRGPRX2 ligands in Siglec-8 transgenic mice induced inflammation and itch, which were inhibited with a Siglec-8 mAb. MRGPRX2-mediated MC activation contributes to inflammation in patients with AD through the release of chemotactic, proteolytic, pruritic and inflammatory factors. Therapeutic strategies that broadly target MCs, such as a lirentelimab (AK002) and a Siglec-8 mAb, may decrease the severity of AD.