The breast gland is composed of epithelial ducts that are undergoing branching morphogenesis resulting in structures called terminal lobular units (TDLUs). The TDLUs are surrounded by vascular-rich stroma. We are focusing on the role of the extracellular matrix (ECM) protein fibronectin (FN). Cells are able to bind to FN through integrins (mainly α5β1) integrated in the cell membrane. Thus, FN is able to transmit extracellular signals into the cell and further induce/regulate the activation of pathways and genes. FN is involved in cell adhesion, differentiation, growth and migration. Here we show the localisation of FN in human mammary glands along with the expression of various other molecules such as CK14, -17, -19, E-, N- and P-Cad, EpCAM, ThyI, Vimentin, Notch2 and Btbd7. To study the role of FN, we established an efficient knockdown of FN in the breast epithelial stem cell line D492. Compared with the untransfected cell line and a scrambled control, we verified a reduction in the expression of variuos markers, such as EpCAM, CK14 and -19, ThyI, P-Cad and Notch2. Further, D492^FNKD cells show a signficantly higher migratory potential and FN knockdown renders the cells more resistant to apoptosis compared to D492^scr. When cultivating these cells, D492^scr and D492^FNKD, respectively, in 3-dimensional Matrigel ECM the resulting branching structures are remarkedly different. D492^FNKD derived structures show signficantly longer ducts compared to D492^scr derived structures.