A series of subclones were isolated from cloned but phenotypically diverse MC3T3-E1 cell lines, and subclones exhibiting either high or low osteoblast differentiation and mineralization were selected from cultures grown in ascorbic acid-containing medium. MC3T3-E1 Subclone 4 and MC3T3-E1 Subclone 14, when grown in the presence of ascorbic acid and 3–4 mM inorganic phosphate, demonstrated a high level of osteoblast differentiation. They formed a well-mineralized extracellular matrix (ECM) after 10 days. In contrast, MC3T3-E1 Subclone 24 and MC3T3-E1 Subclone 30, grown in ascorbic acid, showed poor osteoblast differentiation and did not form an ECM, serving as negative controls for MC3T3-E1 Subclone 4 and MC3T3-E1 Subclone 14. The mineralizing subclones were selected based on their expression of mRNAs for osteoblast markers such as sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone/parathyroid hormone-related protein receptor. Subclones with either high or low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-associated transcription factor. Upon implantation into immunodeficient mice, the highly differentiating subclones formed small bone-like woven bone resembling bone tissue, whereas the poorly differentiating cells produced only fibrous tissue. These cell lines serve as good models for studying osteoblast differentiation in vitro, particularly ECM signaling. Their behavior is similar to that of primary calvarial osteoblast cultures.