Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single‐cell level. Multiparameter flow cytometry permits simultaneous detection of two or more cytokines within a single cell, allowing direct T_H1 versus T_H2 determination. This capability, combined with the high throughput inherent in the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single‐cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell‐surface staining are also described. Because of the greater level of nonspecific binding inherent in fixed, permeabilized cells, greater care must be taken in designing specificity controls. A protocol in which brefeldin A is used to identify cytokine‐producing T cells activated in response to in vivo contact with microbial antigens is also described. Curr. Protoc. Immunol. 78:6.24.1‐6.24.21. © 2007 by John Wiley & Sons, Inc.