AbstractBone defects (BDs), a prevalent clinically refractory orthopaedic disease, presently have no effective treatments. Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and serve as potential seed cells for bone tissue engineering for BD treatment. However, the feasibility of using MSCs as seed cells for bone tissue engineering remains unclear. As a result, the critical issue of large‐scale cell‐scaffold preparation remains unresolved. In this study, we demonstrated for the first time that human embryonic stem cell‐derived MSCs, also known as immunity‐and‐matrix‐regulatory cells (IMRCs), could be inoculated into microcarriers to create osteogenic micro‐tissues appropriate for scalable production in 250 mL bioreactor. IMRCs were generally smaller than umbilical cord‐derived MSCs (UCMSCs) and could attach, migrate, proliferate and differentiate within the porous microcarriers, whereas UCMSCs could only attach to the surface of microcarriers. Osteogenic micro‐tissues generated from IMRCs‐seeded microcarriers significantly increased osteocalcin levels after 21 days of differentiation in a bioreactor. Furthermore, the expression levels of osteogenic biomarker genes/proteins such as alkaline phosphatase (ALP), osteocalcin (OCN), runt‐related transcription factor 2 (RUNX2), osteopontin (OPN) and osterix (OSX) were significantly higher than osteogenic micro‐tissues derived from UCMSCs‐seeded microcarriers. Our findings imply that IMRCs could potentially serve as seed cells for the scalable production of osteogenic micro‐tissues for BD treatment.