A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.