It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.