Poly(ethylene glycol)-b-poly(l-alanine) (PEG-L-PA)s with L-PA molecular weights of 620, 1100, and 2480 Da and a fixed molecular weight of PEG at 5000 Da were synthesized to compare the thermosensitive behavior, and to investigate their potential as a three-dimensional (3D) culture matrix of adipose-tissue-derived stem cells (ADSCs). The sol-to-gel transition temperature and the concentration ranges where the transition was observed decreased as the L-PA molecular weight increased. ADSCs were cultured in the 3D matrixes of in situ formed PEG-L-PA hydrogels, which were produced by increasing the temperature of cell-suspended PEG-L-PA aqueous solutions. The spherical morphology was maintained in the PEG-L-PA hydrogel, while the cells underwent fibroblastic morphological changes in the Matrigel over 14 days of incubation. ADSCs exhibited high expression of type II collagen in the PEG-L-PA thermogel. In addition, they also moderately expressed the biomarker of myogenic differentiation factor 1 as the same mesodermal lineages, as well as the type III β-tubulin as a cross-differentiation biomarker. Similar to the in vitro study, the ADSCs predominantly exhibited chondrogenic biomarkers in the in vivo study. The study demonstrates that the polypeptide thermogel of PEG-L-PA is promising as a 3D culture matrix of ADSCs and as an injectable tissue engineering biomaterial.