Background: Hair follicle stem cells (HFSCs) are promising candidates for cell-based therapies in neurodegenerative diseases because of their ability to differentiate into neural lineages and exert paracrine effects in damaged tissues. However, their clinical application faces challenges, particularly in efficiently guiding them toward neural lineages. This study explores using chick embryo extract (CEE) to enhance HFSCs’ secretory capacity and neuronal differentiation. Materials and Methods: HFSCs from rat whisker pads were cultured in growth medium supplemented with either 20% FBS or a combination of 10% FBS and 10% CEE, transitioning to 20% FBS after the first subculture. We conducted gene expression profiling of lineage commitment markers and neurotrophic factors in both experimental groups, alongside morphological assessments and protein expression analyses. Results: CEE supplementation during migration increased neuronal differentiation, evidenced by more cells with neurites and higher MAP2 expression at both the gene and protein levels. CEE also inhibited the expression of PDGFR-α, indicating a suppression of differentiation toward Schwann cells. Furthermore, we observed increased levels of trophic factors such as BDNF and VEGF at passage 3 induced by CEE supplementation. Conclusions: Enhancing the neuronal lineage commitment of hair follicle stem cells (HFSCs) and boosting the expression of trophic and angiogenic factors through short-term CEE preconditioning during their migratory stage presents a compelling approach. This strategy holds great promise in enhancing the effectiveness of stem cell-based therapies for neurological disorders.