Ectomesenchymal cells isolated from the first branchial arch have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. This study was aimed to confirm the plasticity of multilineage differentiation with molecular and cellular characterization. Monolayer cultures of ectomesenchymal cells harvested from the first branchial arch primordia in embryonic day 9.5 BALB/c mice were passaged 3 times before analysis. Staining with antibodies against S-100, p75 and vimentin suggested that the population of stem cells originated from ectomesenchyme, with few contaminating cells stained for cytokeratin. Then, cells were transferred to adipogenic, osteogenic, chondrogenic and odontogenic media. The initiation of controlled differentiation was determined with histological assays, and the expression of tissue-specific genes was detected using immunocytochemical staining and reverse transcription polymerase chain reaction. The adipogenic ectomesenchymal cells showed accumulation of lipid vacuoles and expression of lipoprotein lipase and peroxisome proliferator-activated receptor γ2. Following osteoinduction, the fibroblast-like cells became cuboidal and formed mineralized nodules. In addition, the expression of mRNA encoding osteocalcin and osteopontin proved osteogenesis at the molecular level. Chondrogenic lineage expressed collagen type II, aggrecan and Sox9 with a low level of collagen type I in monolayer culture. Odontogenesis was determined by dentin sialophosphoprotein, collagen type I and dentin matrix protein 1 expression. Therefore, we have demonstrated that ectomesenchymal cells from the first branchial arch are capable of extensive multilineage differentiation in vitro, controllable by the culture environment. This makes them a relevant and valuable source of stem cells for research of craniofacial development and tissue engineering of restoration.