Reproducible osteogenicity is a key requirement for the clinical use of bone substitutes based on bone marrow stromal cells (BMSCs) and three-dimensional (3D) scaffolds. In this study we addressed whether a minimal cell density is required for ectopic osteogenicity of constructs generated using a recently developed perfusion system for seeding and culturing human BMSCs on 3D scaffolds. Cells from human bone marrow aspirates were directly seeded and expanded for 3 weeks within the pores of ceramic-based scaffolds, using a perfusion bioreactor. The resulting constructs were either implanted subcutaneously in nude mice, to determine their capacity to generate bone tissue, or digested to retrieve the expanded cells and assess their number, phenotype and clonogenic capacity. The final number of BMSCs in the constructs was correlated neither to the initial number of seeded cells, nor to the subsequent bone formation. Instead, the final number of clonogenic BMSCs in the constructs was positively correlated to the initial number of BMSCs seeded, and was significantly higher in osteogenic than in non-osteogenic constructs. These results indicate that clonogenic cells play a crucial role in determining the osteogenicity of engineered bone substitutes. Possible ways to quantify the density of clonogenic cells as a quality control parameter to predict potency of BMSC-based constructs are discussed. Copyright © 2007 John Wiley & Sons, Ltd.