2D/3D hybrid cell culture systems are constructed by increasing the temperature of the thermogelling poly(ethylene glycol)-poly(L-alanine) diblock copolymer (PEG-L-PA) aqueous solution in which tonsil tissue-derived mesenchymal stem cells and graphene oxide (GO) or reduced graphene oxide (rGO) are suspended, to 37 degrees C. The cells exhibit spherical cell morphologies in 2D/3D hybrid culture systems of GO/PEG-L-PA and rGO/PEG-L-PA by using the growth medium. The cell proliferations are 30%-50% higher in the rGO/PEG-L-PA hybrid system than in the GO/PEG-L-PA hybrid system. When chondrogenic culture media enriched with TGF-beta 3 is used in the 2D/3D hybrid systems, cells extensively aggregate, and the expression of chondrogenic biomarkers of SOX 9, COL II A1, COL II, and COL X significantly increases in the GO/PEG-L-PA 2D/3D hybrid system as compared with the PEG-L-PA 3D systems and rGO/PEG-L-PA 2D/3D hybrid system, suggesting that the GO/PEG-L-PA 2D/3D hybrid system can be an excellent candidate as a chondrogenic differentiation platform of the stem cell. This paper also suggests that a 2D/3D hybrid system prepared by incorporating 2D materials with various surface biofunctionalities in the in situ forming 3D hydrogel matrix can be a new cell culture system.