The purposes of this study were to quantify the number of replicating mesenchymal cells and to correlate it with the amount of new bone formed in the glenoid fossa during stepwise advancement. We randomly divided 250 female Sprague-Dawley rats, 35 days old, into 10 control groups (n = 5) and 20 experimental groups (n = 10). Fifty rats from the stepwise experimental group received initial advancement of 2 mm and another 1.5 mm of advancement on day 30 by the addition of veeners. On days 3, 7, 14, 21, 30, 33, 37, 44, 51, and 60, the rats were killed. One hour before that, the rats were injected with bromodeoxyuridine (BrdU) intravenously. We cut 7-microm tissue sections through the glenoid fossa sagittally and stained them with anti-BrdU antibody to evaluate the number of replicating mesenchymal cells. During the first advancement, the number of replicating cells in the posterior region of the glenoid fossa showed a significant increase compared with natural growth, but a significant decrease compared with 1-step advancement. On the second advancement, however, an increase in the number of replicating cells was observed on day 37 with a subsequent and significant increase in bone formation on day 44. Mandibular advancement conducted in a stepwise fashion increases the number of replicating mesenchymal cells in the glenoid fossa. However, a minimum threshold of strain must first be exceeded before these mesenchymal cells can differentiate to ultimately form new bone.