Background: Mast cells produce a variety of cytokines and chemokines in a timely and tightly controlled fashion if stimulated via the FcεRI. Evidence is accumulating that the transcriptional induction of the corresponding genes and the release of these mediators are dependent on common and mediator–specific components of the signal transduction and transcription factor machinery. Methods: We addressed this issue by comparing the effects of mitogen activated protein (MAP) kinase pathway inhibitors and protein kinase C (PKC) inhibitors on the induction of TNF–α and IL–5 after IgE plus antigen (Ag) stimulation in CPII mouse mast cells using Western blot analyses and transient transfections of reporter gene plasmids. Results: TNF–α shows a strict dependence on the MAP kinase pathway, while IL–5 is either activated by PMA–dependent PKCs or along the MAP kinase pathway. In addition, both mediators are sensitive to PKCμ inhibition, suggesting involvement of this atypical, non–PMA dependent PKC in the overall induction process. Conclusion: While the two cytokines were recently shown to be regulated by a member of the nuclear factor of activated T–cells (NF–AT) transcription factor family, activator protein 1 (AP1) was identified as a cofactor at the TNF–α promoter while a GATA family member comprised the cofactor at the IL–5 promoter. This suggests that the differences in requirement for signal transduction cascades are the result of a different usage of NF–AT cofactors for transcription of each cytokine in mast cells.