The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs‐based tissue‐engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose‐derived mesenchymal stromal cells (ADMSCs) in three‐dimensional (3D) cocultures. For this, we cocultured THP‐1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic‐co‐glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP‐2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt‐related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro‐environment in influencing cell‐based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment.

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