The authors browsed through past genetic findings in hemifacial microsomia along with our previously identified mutations in ITGB4 and PDE4DIP from whole genome sequencing of hemifacial microsomia patients. Wondering whether these genes influence mandibular bone modeling by regulation on osteogenesis, the authors approached mechanisms of hemifacial microsomia through this investigation into gene knockdown effects in vitro. MC3T3E1 cells were divided into 5 groups: the negative control group without osteogenesis induction or siRNA, the positive control group with only osteogenesis induction, and 3 gene silenced groups with both osteogenesis induction and siRNA. Validation of transfection was through fluorescence microscopy and quantitative real-time Polymerase chain reaction on knockdown efficiency. Changes in expression levels of the 3 genes during osteogenesis and impact of Itgb4 and Pde4dip knockdown on osteogenesis were examined by quantitative real-time Polymerase chain reaction, alkaline phosphatase, and alizarin red staining. Elevation of osteogenic genes Alpl, Col1a1, Bglap, Spp1, and Runx2 verified successful osteogenesis. Both genes were upregulated under osteogenic induction, while they had different trends over time. Intracellular fluorophores under microscope validated successful transfection and si-m-Itgb4_003, si-m-Pde4dip_002 had satisfactory knockdown effects. During osteogenesis, Pde4dip knockdown enhanced Spp1 expression (1.95 ± 0.13 folds, P = 0.045). The authors speculated that these genes may have different involvements in osteogenesis. Stimulated expression of Spp1 by Pde4dip knockdown may suggest that Pde4dip inhibits osteogenesis.