In the application of adult stem cells in regenerative medicine, it is indispensable to control stem cell behaviour in vitro. Since stem cells spontaneously differentiate into several cell types, it is mandatory to identify methods to enrich the desired cell types and concurrently block other differentiation pathways. More precisely, generation of a defined cell population is a key prerequisite for a therapeutic application of stem cells. Here we have demonstrated that it is possible to influence the differentiation of human pancreatic stem cells (hPSCs). During activation of mesodermal differentiation, the cytoskeletal protein alpha-smooth muscle actin (α-SMA) seems to play an important role in different cell systems and can usually be detected in hPSCs during in vitro cultivation. We cultured stem cells under different conditions and analyzed the impact on α-SMA expression. On the one hand, supplements like retinoic acid (RA) and dimethyl sulfoxide (DMSO) were added to the cultivation medium; on the other hand, different media with or without the addition of fetal calf serum (FCS) were used. Expression of α-SMA was determined by immunocytochemistry, Western blot analysis and quantitative RT-PCR. After the treatment of hPSCs with RA, a strong induction of α-SMA protein expression was observed when 2 mM RA was added to the medium. DMSO in turn induced a marked reduction in α-SMA-positive cells. This could also be observed using a keratinocyte serum-free medium (KSFM). Furthermore, the general addition of FCS to the medium had a blocking effect on α-SMA expression and decreased the number of α-SMA-positive cells to a minimum. The controlled modulation of hPSCs by soluble factors is a first success on the way to a promising application for transplantation medicine and cell therapy of degenerative diseases.