The presentation of TGF-β1 during the early stage of wound healing is a prerequisite for extracellular matrix (ECM) synthesis and remodeling by activated fibroblasts, called myofibroblasts. At later stages, clearance of myofibroblasts is needed to avoid overshooting ECM production. Apoptosis of myofibroblasts and the macrophage-released anti-inflammatory cytokine IL-10 are controversially discussed as regulating cues in this context. To reveal the regulating cues, defined biomaterial scaffolds are needed to conduct in-depth in vitro studies in a physiologically relevant context. In this work, we used an in vitro biomimetic wound healing model. It consists of a 3D fibrillar matrix from collagen I and fibronectin and different temporal stimuli by TGF-β1 and IL-10. Human dermal fibroblast behavior was investigated in terms of myofibroblast differentiation (αSMA expression), matrix remodeling, proliferation and migration in the permanent or sequential presence of TGF-β1 and IL-10 over 4 days. We could show that removal of TGF-β1 after initial stimulation resulted in an increase of apoptosis of myofibroblasts. In contrast, TGF-β1 stimulation followed by IL-10 treatment did not result in increased cell apoptosis but instead led to a significant increase of cell motility and reduction of myofibroblasts. The findings suggest that myofibroblasts are a transiently "activated" fibroblastic phenotype and can be de-differentiated to fibroblasts in the presence of IL-10. Overall, our 3D ECM model allows mimicking the early and late stages of wound healing and highlights the temporal sequence of TGF-β1 and IL-10 as an important cue for completion of tissue formation and maintenance of tissue homeostasis.