β2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of β2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2^−/−), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2^+/+) and Adrb2^−/− mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2^+/+ and Adrb2^−/− mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2^+/+ UAC mice;​ the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression;​ the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA.