Transfusion-related　lung　injury　(TRALI)　poses　a　significant　risk　following　blood　transfusion　and　remains　the　primary　cause　of　transfusion-related　morbidity　and　mortality,　primarily　driven　by　the　activation　of　immune　cells　through　anti-major　histocompatibility　complex　class　I　(anti-MHC　I)　antibody.　However,　it　remains　to　be　defined　how　immune　microenvironmental　cue　contributes　to　TRALI.　Here,　we　uncover　that　activated　mast　cells　within　the　immune　microenvironment　promote　lung　inflammation　and　injury　in　antibody-mediated　TRALI,　both　in　vitro　and　in　vivo.　This　was　demonstrated　by　co-culturing　lipopolysaccharide　(LPS)-pretreated　mast　cell　line　with　anti-MHC　I　antibody　and　establishing　a　“two-hit”　TRALI　mouse　model　through　intratracheal　injection　of　LPS　followed　by　tail-vein　injection　of　anti-MHC　I　antibody.　Importantly,　mast　cell-deficient　KitW-sh/W-sh　mice　exhibited　markedly　reduced　lung　inflammation　and　injury　responses　in　antibody-mediated　TRALI　compared　with　wild-type　mice.　Mechanistically,　activation　of　toll-like　receptor　3　(TLR3)/mitogen-activated　protein　kinase　(MAPK)　signaling　pathway　in　mast　cells　contributes　to　the　enhanced　production　of　proinflammatory　factors.　These　excessive　proinflammatory　factors　produced　by　activated　mast　cells　contribute　to　lung　inflammation　and　injury　in　antibody-mediated　TRALI.　Pharmacologically　targeting　the　TLR3/MAPK　pathway　to　inhibit　mast　cell　activation　normalizes　the　proinflammatory　microenvironment　and　alleviates　lung　inflammation　and　injury　in　the　preclinical　TRALI　mouse　model.　Overall,　we　find　that　activation　of　mast　cells　via　the　TLR3/MAPK　pathway　contributes　to　lung　inflammation　and　injury　in　antibody-mediated　TRALI,　providing　novel　insights　into　its　underlying　mechanisms.　Furthermore,　targeting　activated　mast　cells　and　the　associated　pathway　offers　potential　therapeutic　strategies　for　antibody-mediated　TRALI.　©　2024　The　Authors