Poly(ethylene glycol)-poly(l-alanine) diblock copolymer (PEG-L-PA; molecular weight of each block of 1000-1080 Da) aqueous solutions undergo sol-to-gel transition in a 3.0-8.0 wt % concentration range as the temperature increases. By incorporating the polystyrene microspheres with different functional groups with a size of 100-800 μm in in situ formed PEG-L-PA thermogels, the differentiation of tonsil-tissue-derived mesenchymal stem cells (TMSCs) was investigated. The mRNA expression and immunohistochemical assays suggested that the TMSCs preferentially undergo adipogenesis in the ammonium (-NH3(+))- or thiol (-SH)-functionalized microsphere incorporated thermogels; chondrogenesis in the thiol-, phosphate (PO3(2-))-, or carboxylate (-COO(-))-functionalized microsphere incorporated thermogels; and osteogenesis in the phosphate-, carboxylate-functionalized, or neat polystyrene microsphere incorporated thermogels. This paper provides a new TMSC 3D culture system of a sol-gel reversible matrix and suggests that the surface-functional groups of microspheres in the thermogel can control the preferential differentiation of stem cells into specific cell types during the 3D culture.