Establishing the molecular and cellular mechanisms of fibrosis requires the development of validated and reproducible models. The complexity of in vivo models challenges the monitoring of an individual cell fate, in some cases making it impossible. However, the set of factors affecting cells in in vitro culture systems differ significantly from in vivo conditions, insufficiently reproducing living systems. Thus, to model profibrotic conditions in vitro , usually the key profibrotic factor, transforming growth factor beta (TGFβ-1) is used as a single factor. TGFβ-1 stimulates the differentiation of fibroblasts into myofibroblasts, the main effector cells promoting the development and progression of fibrosis. However, except for soluble factors, the rigidity and composition of the extracellular matrix (ECM) play a critical role in the differentiation process. To develop the model of more complex profibrotic microenvironment in vitro , we used a combination of factors: decellularized ECM synthesized by human dermal fibroblasts in the presence of ascorbic acid if cultured as cell sheets and recombinant TGFβ-1 as a supplement. When culturing human mesenchymal stromal cells derived from adipose tissue (MSCs) under described conditions, we observed differentiation of MSCs into myofibroblasts due to increased number of cells with stress fibrils with alpha-smooth muscle actin (αSMA), and increased expression of myofibroblast marker genes such as collagen I, EDA-fibronectin and αSMA. Importantly, secretome of MSCs changed in these profibrotic microenvironment: the secretion of the profibrotic proteins SPARC and fibulin-2 increased, while the secretion of the antifibrotic hepatocyte growth factor (HGF) decreased. Analysis of transciptomic pattern of regulatory microRNAs in MSCs revealed 49 miRNAs with increased expression and 3 miRNAs with decreased expression under profibrotic stimuli. Bioinformatics analysis confirmed that at least 184 gene targets of the differently expressed miRNAs genes were associated with fibrosis. To further validate the developed model of profibrotic microenvironment, we cultured human dermal fibroblasts in these conditions and observed increased expression of fibroblast activation protein (FAPa) after 12 hours of cultivation as well as increased level of αSMA and higher number of αSMA+ stress fibrils after 72 hours. The data obtained allow us to conclude that the conditions formed by the combination of profibrotic ECM and TGFβ-1 provide a complex profibrotic microenvironment in vitro . Thus, this model can be applicable in studying the mechanism of fibrosis development, as well as for the development of antifibrotic therapy.