Studies of mice rendered deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) have established unique roles for these cytokines in pulmonary homeostasis, resistance to infection, and antigen-specific T- and B-cell responses. In addition to these distinctive properties, however, GM-CSF and IL-3 also stimulate the development and activation of hematopoietic cells in many similar ways, raising the possibility that each factor might partially compensate for the other's absence in singly deficient mice. To test whether endogenous GM-CSF and IL-3 mediate redundant functions in vivo, we generated mice lacking both cytokines through sequential gene targeting experiments in embryonic stem (ES) cells. Surprisingly, doubly deficient animals, but not single knockouts, showed increased numbers of circulating eosinophils. Doubly deficient mice, moreover, developed weaker contact hypersensitivity reactions to haptens applied epicutaneously than mice deficient in either factor alone. Together, these findings delineate overlapping roles for GM-CSF and IL-3 in hematopoiesis and immunity. (Blood. 2001;97:922-928)