Mast cell development, function, and survival are likely to be regulated by a complex interplay of cellular signaling. Usually, these signals derive from the cellular milieu associated with the specific mast cell environment in health or disease conditions. A major methodological issue in studying in vitro mast cells, as well as any other tissue dwelling cell, is the essential lack of all the tissue-derived signals. Because some of the signals can be unknown, the in vivo system they form is virtually impossible to mimic completely in vitro. The mast cell-fibroblast co-culture system partially overcomes this problem and is the main topic of this chapter. The experimental importance of mast cell-fibroblast co-culture for the mast cells derives mainly from two reasons: first, fibroblasts constitute a major cellular scaffold of the tissues where mast cells dwell in the body, and as such are one of the fundamental cells participating in mast cell regulation in vivo and, second, there is an analogy with the traditional model of allergic inflammation, where the late, chronic phase is characterized by mast cell-structural cell crosstalk and eventual fibrotic outcome. Therefore, the co-culture system can be viewed as a suitable tool to investigate mast cell-fibroblast crosstalk.