Background: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable, high-throughput assays limits the clinical utility of this approach. The aim of this study was to develop a high-throughput Basophil Activation Test (BAT), based on human progenitor cell-derived basophils (PCB), and to investigate the role of PCB activation test (PCBAT) in allergy diagnosis. Methods: PCBs were differentiated from CD34+ progenitor cells, and sensitized with sera from subjects sensitized to cat (n=35, 17 subjects clinical reactivity validated), peanut-allergic (n=30, 15 subjects clinical reactivity validated), peanut-sensitized but tolerant subjects (n=13). Sensitized PCBs were then stimulated with a range of concentrations of the corresponding allergens and degranulation was measured using CD63 expression on flow cytometry. Results: Following passive sensitisation of the mature PCB (2D7+/FcεRI+/CD117-/HLADR-) with serum and stimulation with allergen, we saw a dose-dependent increase in CD63 expression which was allergen specific. In subjects sensititsed to cat there was a positive correlation between PCBAT area under curve (AUC) versus specific IgE (sIgE) to cat (p=0.001) and versus airway responsiveness to inhaled cat allergen (p=0.026). There was a significant negative correlation between PCBAT AUC for peanut allergen and response to oral food challenge test to peanut - subjects with higher PCBAT AUC reacted to a lower dose on the oral food challenge to peanut (p=0.001), and had higher sIgE to Ara h 1 (p=0.007). All peanut tolerant subjects showed no reaction to peanut on PCBAT. Conclusion: PCBAT may confer a powerful alternative tool in allergy testing.