AbstractDirected differentiation of induced pluripotent stem cells (iPSCs) towards specific lineages remains a major challenge in regenerative medicine, while there is a growing perception that this process can be influenced by the three-dimensional environment. In this study, we investigated whether iPSCs can differentiate towards mesenchymal stromal cells (MSCs) when embedded into fibrin hydrogels to enable a one-step differentiation procedure within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like phenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, faint expression of CD73 and CD105, and reduced differentiation potential towards osteogenic and adipogenic lineage. Transcriptomic analysis demonstrated that characteristic genes for MSCs and extracellular matrix were upregulated on flat substrates, whereas genes of neural development were upregulated in 3D culture. Furthermore, the 3D culture had major effects on DNA methylation profiles, particularly within genes for neuronal and cardiovascular development, while there was no evidence for epigenetic maturation towards MSCs. Taken together, iPSCs could be differentiated towards MSCs on tissue culture plastic or on a flat fibrin hydrogel. In contrast, the differentiation process was heterogeneous and not directed towards MSCs when iPSCs were embedded into the hydrogel.