A one-step concept for bone regeneration has been postulated in which human adipose stem cells (hASCs) are harvested, triggered to differentiate, seeded on carriers, and implanted in the same operative procedure. Toward this goal it was investigated whether short (minutes) incubation with bone morphogenetic protein-2 (BMP-2) suffices to trigger osteogenic differentiation of hASCs seeded on calcium phosphate carriers. hASCs were treated with or without BMP-2 (10 ng/mL) for 15 min, and seeded on β-tricalcium phosphate granules (β-TCP; sized <0.7 mm or >0.7 mm) or biphasic calcium phosphate (BCP; 60%/40% or 20%/80% hydroxyapatite/β-TCP). Attachment was determined after 10-30 min. Proliferation (DNA content) and osteogenic differentiation (alkaline phosphatase activity, gene expression) were analyzed up to 3 weeks of culture. hASC attachment to the different scaffolds was similar, and unaffected by BMP-2. It stimulated gene expression of the osteogenic markers core binding factor alpha 1, collagen-1, osteonectin, and osteocalcin in hASCs seeded on BCP and β-TCP. Downregulation of osteopontin expression by BMP-2 was seen in BCP-seeded cells only. BMP-2 treatment inhibited expression of the adipogenic marker peroxisome proliferator-activated receptor gamma. In conclusion, 15 min BMP-2 preincubation of hASCs seeded on BCP/β-TCP scaffolds had a long-lasting stimulating effect on osteogenic differentiation in vitro. These results strongly support a one-step clinical concept for bone regeneration.