Synovial fibroblasts have attracted considerable attention in studies of joint diseases. Although mice are useful and powerful in in vitro and in vivo experiments, primary cultures of mouse synovial fibroblasts are notoriously difficult because the mouse synovial tissues are much smaller and cell cycle arrests can be induced more easily in murine cells than in human cells. Here, we report a precise protocol for the isolation and culture of fibroblasts from mouse adipose and fibrous knee joint synovia. In both adipose and fibrous synovial fibroblasts, proliferation was decreased in addition to a higher rate of cellular senescence under normoxic conditions (20% O); however, it was maintained over 20 days with low cellular senescence under hypoxic conditions (3% O). The marker gene expression in adipose and fibrous synovial fibroblasts was not markedly altered after a 3-week culture. Both cells displayed similar potencies for chondrogenic, osteogenic, and adipogenic differentiation, and responses to a proinflammatory cytokine. The present method provides a sufficient amount of mouse synovial fibroblasts for in vitro and in vivo experiments in joint biology and the pathophysiology of osteoarthritis and rheumatoid arthritis.