Objective. We previously reported that human synovium contains cells that, after culture expansion, display properties of mesenchymal stem cells (MSCs). The objective of this study was to identify MSCs in native synovium in vivo.Methods. To identify stem cells in the synovium in vivo, a double nucleoside analog cell-labeling scheme was used in a mouse model of joint-surface injury. For labeling of slow-cycling cells, mice received iododeoxyuridine (IdU) for 30 days, followed by a 40-day washout period. For labeling of cells that proliferate after injury, mice underwent knee surgery to produce an articular cartilage defect and received chlorodeoxyuridine (CIdU) for 4 days, starting at multiple time points after surgery. Unoperated and sham-operated joints served as controls. Knee joint paraffin sections were analyzed by double and triple immunostaining to detect nucleoside analogs, conventional MSC markers, and chondrocytelineage markers.Results. Long-term-retaining, slow-cycling IdUpositive cells were detected in the synovium. At 4 days and 8 days after injury, there was marked proliferation of IdU-positive cells, which costained for CIdU. IdUpositive cells were nonhematopoietic, nonendothelial stromal cells, were distinct from pericytes, and stained positive for MSC markers. MSCs were phenotypically heterogeneous and located in topographically distinct niches in the lining layer and the subsynovial tissue. Twelve days after injury, double nucleoside-labeled cells within synovium were embedded in cartilagespecific metachromatic extracellular matrix and costained positive for the chondrocyte-lineage markers Sox9 and type II collagen.Conclusion. Our findings provide the first evidence of the existence of resident MSCs in the knee joint synovium that undergo proliferation and chondrogenic differentiation following injury in vivo.