Rationale: Eosinophils develop from stem cells in the bone marrow under the influence of hematopoietic cytokines, particularly IL-5. Previously, we have demonstrated that blockage of Notch signaling by a -secretase inhibitor (GSI) promotes the differentiation of umbilical cord blood (UCB)-derived eosinophils. These highly major basic protein (MBP)-positive eosinophils cultured in the presence of inhibitor lack the migratory response to eotaxin, although their CCR3 levels are similar to those of eosinophils cultured without inhibitor. Objectives: We investigated the mechanism underlying the differential responses of differentiating eosinophils and their functionalities in response to eosinophil-active cytokines in the presence and absence of GSI. Methods: UCB cells cultured for 4 weeks with hematopoietic cytokines in the presence or absence of GSI were monitored for ERK phosphorylation, MBP expression, and functionality. Measurements and Main Results: Eosinophil differentiation from UCB cells was accompanied by activation of the ERK1/2 pathway during the 4-week culture period. In particular, strong ERK1/2 phosphorylation was observed in eosinophils during the final stage of culture when GSI was present. Consistent with this finding, ERK inhibition nullified the effect of GSI on eosinophil differentiation. Eosinophils cultured with GSI resembled airway eosinophils rather than peripheral blood eosinophils based on reduced IL-5R expression, blunted eosinophil cationic protein (ECP) degranulation, and decreased IL-13 and GM-CSF production. Conclusions: These results suggest that Notch signaling regulates the terminal differentiation and subsequent effector phenotypes of eosinophils, partly through modulation of the ERK pathway. GSI has therapeutic potential for eosinophilic inflammatory diseases, such as asthma.