Microenvironmental factors such as biomaterial physical and chemical properties, mechanical stimuli, and soluble chemical cues integrate in vivo to direct the differentiation of mesenchymal stromal cells (MSCs). Identifying what factor combinations best promote the differentiation of MSCs into myofibroblasts (tissue-synthetic cells of developing valves), but not into osteoblasts, is challenging when designing for heart valve tissue engineering (HVTE). To date, select combinations of microenvironmental factors have been studied in two-dimensional (2D) culture, but systematic consideration of the integration of factors in 3D culture has not been explored fully. We first developed a screening platform using polyethylene glycol norbornene (PEG-NB) as a model biomaterial with which the polymer wt% (to control elastic modulus), adhesion peptide types (RGD, DGEA, YIGSR) and densities, and soluble TGF-β1 could be controlled independently and combinatorially in arrays of 3D hydrogels. We then utilized a deformable membrane platform to screen those same factors under dynamic mechanical stimulation. In a static system, low PEG-NB wt% materials with high concentrations of RGD permitted significant spreading and expression of myofibroblastic markers (α-SMA and collagen type I) in the presence of TGF-β1. In the dynamic system, multiple metrics for α-SMA and collagen type I expression converged to show that mechanical stimulation and TGF-β1 can have different individual and integrated effects on myofibrogenesis depending on the presence of other factors (most notably, PEGNB or RGD), meaning that design decisions could be erroneous if made based on experiments that do not consider integrative effects. When combined with a design of experiments approach, statistical modelling defined previously undescribed response relationships that predicted conditions (subsequently experimentally validated) that promoted maximal α-SMA and collagen expression beyond the initial screening data. Modelling complex interactions has the potential to reveal key regulatory pathways for myofibrogenesis, which has implications for study in other areas like pathological fibrosis and cancer research.