Osteoblasts are responsible for the maintenance of bone homeostasis. Deregulation of their differentiation is etiologically linked to several bone disorders, making this process an important target for therapeutic intervention. Systemic identification of osteoblast regulators has been hampered by the unavailability of physiologically relevant in vitro systems suitable for efficient RNAi and for differentiation read-outs compatible with fluorescent microscopy-based high-content analysis (HCA). Here, we report a new method for identification of osteoblast differentiation regulators by combining siRNA transfection in physiologically relevant cells with high-throughput screening (HTS). Primary mouse calvarial osteoblasts were seeded in 384-well format and reverse transfected with siRNAs and their cell number and differentiation was assayed by HCA. Automated image acquisition allowed high-throughput analyses and classification of single cell features. The physiological relevance, reproducibility, and sensitivity of the method were validated using known regulators of osteoblast differentiation. The application of HCA to siRNAs against expression of 320 genes led to the identification of five potential suppressors and 60 activators of early osteoblast differentiation. The described method and the associated analysis pipeline are not restricted to RNAi-based screening, but can be adapted to large-scale drug HTS or to small-scale targeted experiments, to identify new critical factors important for early osteoblastogenesis.