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Background: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high‐throughput assays limits the clinical utility. We aimed to develop a high‐throughput basophil activation test based on human progenitor cell‐derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases. Methods: Progenitor cell‐derived basophils were differentiated from CD34+ progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed. Results: Following passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose‐dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut‐sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r2 =.49, p =.001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC20) correlated with PCBAT AUC (r2 =.33, p =.016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r2 =.59, p <.0001). Peanut threshold cumulative dose during DBPCFC was negatively correlated with PCBAT AUC (r2 =.57, p =.001) and IgE to Ara h1 (r2 =.55, p =.007), but not with sIgE to whole peanut or Ara h2. All peanut‐sensitized but tolerant subjects showed no reaction to peanut on PCBAT. Conclusion: Progenitor cell‐derived basophils activation test is a high‐throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing. [ABSTRACT FROM AUTHOR]