Eosinophils are bone marrow-derived cells that differentiate in the bone marrow and migrate into the peripheral blood primarily under the regulation of interleukin (IL)-5. Eosinophil levels in the blood are relatively low. However, under steady-state conditions and in settings of allergic inflammation, parasite infections, or even cancer, they migrate and mainly reside in mucosal tissues where they have key effector and immune-modulating functions. Functional studies using eosinophils are not simple, since these cells are terminally differentiated and rapidly die in vitro. Thus, establishing simple methods to characterize, obtain, and functionally assess eosinophil activities is important. In this unit, we describe methodology for identifying tissue eosinophils by flow cytometry. In addition, we provide detailed methods for isolating eosinophils and for differentiating them from bone marrow cells for further functional studies. © 2017 by John Wiley & Sons, Inc.