Mediators released from activated mast cells are responsible for the allergic inflammatory reactions associated with disease states such as anaphylaxis and atopy. These mediators are released as a consequence of immediate degranulation and phospholipid metabolism upon mast cell activation, followed by delayed cytokine gene expression. Thus, techniques that monitor indices of these events in mast cell culture systems, in association with biochemical analysis of parameters of cell signaling, are critical to our understanding of the molecular mechanisms regulating mast cell–mediated disease. Furthermore, these systems can be adapted for high‐throughput screens to identify potential inhibitors of mast cell activation that may provide potential leads for novel therapies for these diseases. In this unit, we describe approaches that can be readily used or adapted to a variety of rodent and human mast cell culture systems for the determination of degranulation, phospholipid‐derived inflammatory mediator production, and cytokine generation. Curr. Protoc. Immunol. 91:7.38.1‐7.38.9. © 2010 by John Wiley & Sons, Inc.