Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) serve as a promising source for cell-based therapies in regenerative medicine. However, optimal methods for transforming iPSCs into MSCs and the characteristics of iPSC-MSCs obtained from different methods remain poorly understood. In this study, we developed a one-step method for obtaining iPSC-MSCs (CD146+STRO-1+ MSCs) from human synovial fluid MSC-derived induced iPSCs (SFMSC-iPSCs). CD146-STRO-1-SFMSCs were reprogrammed into iPSCs by transduction with lentivirus-mediated Sox2, Oct-3/4, klf4, and c-Myc. SFMSC-iPSCs were maintained with mTeSR1 medium in Matrigel-coated culture plates. Single dissociated cells were obtained by digesting the SFMSC-iPSCs with trypsin. The dissociated cells were then plated into Matrigel-coated culture plate with alpha minimum essential medium supplemented with 10% fetal bovine serum, 1× Glutamax, and the ROCK inhibitor Y-27632. Cells were then passaged in standard cell culture plates with alpha minimum essential medium supplemented with 10% fetal bovine serum and 1× Glutamax. After passaging in vitro, the cells showed a homogenous spindle-shape similar to their ancestor cells (SFMSCs), but with more robust proliferative activity. Flow cytometric analysis revealed typical MSC surface markers, including expression of CD73, CD90, CD105, and CD44 and lack of CD45, CD34, CD11b, CD19, and HLA-DR. However, these cells were positive for CD146 and stro-1, which the ancestor cells were not. Moreover, the cells could also be induced to differentiate in osteogenic, chondrogenic, and adipogenic lineages in vitro. The differentiation potential was improved compared with the ancestor cells in vitro. The cells were not found to exhibit oncogenicity in vivo. Therefore, the method presented herein facilitated the generation of STRO-1+CD146+ MSCs from SFMSC-iPSCs exhibiting enhanced proliferation and differentiation potential.