Key Points• ROSA KIT WT is a new human SCF-dependent FceRI-positive mast cell line that converts to SCF-independence by KIT D816V-transfection.• The FceRI-positive ROSA KIT D816V clone is a major tool for studying cellular aspects of mastocytosis and responses to targeted drugs.In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA -derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA KIT D816V may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients. (Blood. 2014;124(1):111-120)