Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGF) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGF induces expression of smooth muscle alpha actin (SMA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGF induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGF-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGF-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK -/- MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK-/- MEFs. We conclude that FAK does not contribute to TGF-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SMA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.