Migration of human dermal fibroblasts (HDFs) is critical for skin wound healing. The mechanism remains unclear. We report here that platelet-derived growth factor-BB (PDGF-BB) is the major promotility factor in human serum for HDF motility on type I collagen. PDGF-BB recapitulates the full promotility activity of human serum and anti-PDGF neutralizing antibodies completely block it. Although collagen matrix initiates HDF migration without growth factors, PDGF-BB-stimulated migration depends upon attachment of the cells to a collagen matrix. The PDGF-BB's role is to provide directionality and further enhancement for the collagen-initiated HDF motility. To study the collagen and PDGF-BB "dual signaling" in primary HDF, we establish "gene cassettes" plus lentiviral gene delivery approach, in which groups of genes are studied individually or in combination for their roles in HDF migration. Focal adhesion kinase, p21(Rac,CDC42)-activated kinase and Akt are grouped into an upstream kinase gene cassette, and the four major mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 5) are grouped into a downstream kinase gene cassette. The experiments demonstrate 1) the genes' individual roles and specificities, 2) their combined effects and sufficiency, and 3) the mechanisms of their intermolecular connections in HDF migration driven by collagen and PDGF-BB.