Introduction: Smooth muscle cells (SMCs) play an important role in maintaining vascular homeostasis and the development of diseases. We assess feasibility and efficacy of differentiation human induced pluripotent stem cells (hiPSCs) to SMCs in a 3-dimension (3D). Methods: hiPSCs were cultured in shaker flasks or PBS-0.1 Vessels for 48 h. Differentiation initiated (as day 0) by culturing hiPSC spheroids in RPMI+B27 without insulin (B27-) and CHIR99021 (CHIR) for 24 h. Day 1, medium was replaced with RPMI+B27-, platelet derived growth factor β (PDGF-β), and transforming growth factor β1 (TGFβ1) for 48 h, refreshed medium on day 3 for 48 h. Day 5, differentiating hiPSC-spheroids were dissociated to single cells and cultured as monolayer in RPMI +4% FBS, PDGF-β, and TGFβ1 for 72 h. Refreshed medium day 8 for 72 h. Differentiation efficiency was determined by flow cytometry and fluorescence immunostaining. The biological function of 3D differentiated SMCs was assessed in vitro and in a mouse model of ischemic limb disease in vivo . Results: Both 3D differentiation methods efficiently differentiated hiPSCs into SMCs. Flow cytometry showed that 99.4% cells expressed smooth muscle actin (SMA), 97.9% expressed myosin heavy chain 11 (MYH11), 95.1% expressed SM22, 97.5% expressed calponin, and 97.7% expressed calveolin-3 using shaker flasks. They were 99.6% for SMA, 97.9% for MYH11, 97% for SM22, 89.6% for calponin, and 87.2% for calveolin-3, respectively, using PBS-0.1 Vessels. Mean SMC cell number obtained was 4.56 x 10 7 for shaker flasks and 5.69 x 10 7 for PBS-0.1 Vessels, respectively, which were significantly higher than hiPSCs differentiated in monolayer (5.7x10 6 ). Fluorescence immunostaining confirmed that SMCs differentiated from both methods expressed SMA, MYH11, SM22, Calponin, and calveolin-3. Differentiated SMCs migrated in scratch assay, contracted in response to U-46619, an analog of the endoperoxide prostaglandin, and formed tubular structure on Matrigel in vitro. Fluorescence angiogram imaging showed that SMC transplantation effectively induced perfusion in mouse ischemic limbs. Conclusions: Both 3D differentiation methods, using shaker flasks or PBS-0.1 vessels, efficiently differentiated hiPSCs into large scales of functional SMCs.