Engineering hair follicles using hair follicle stem cells (HFSC) is hindered by the low availability of cell source and easy loss of cell phenotype upon in vitro culture. Neonatal human epidermal keratinocytes (HEKn), an alternative cell source to HFSCs, have been employed to recreate in vitro hair follicle by differentiating to hair lineage upon exposure to appropriate inductive signals. However, factors that drive such epithelial cells to hair lineage differentiation have not been fully elucidated. A well‐ established Multiphoton Microfabrication and Micropatterning (MMM) platform enables the fabrication of arbitrary 3D protein microstructures with surface functionalization of niche factors (e.g., extracellular matrix (ECM) proteins, soluble factors) at the micron scale. Herein, we developed a cell niche factor biochip (CNFB) using such technology to screen the individual or combinational niche factor(s) favoring the hair lineage differentiation of HEKn. Specifically, different cell niches including ECM proteins, soluble factors, topological features, and elastic modulus were engineered on the glass surface in each biochip well. After culturing HEKn for 48 hours, the levels of hair lineage differentiation and epidermal stemness were evaluated by immunofluorescence staining of Krt75 and Cytokeratin 5 (C5), respectively. Results showed that fibronectin and BMP2 significantly enhanced Krt75 while reduced C5 expression, demonstrating their positive roles in inducing hair differentiation. Investigating the effect of combinational niche factors in different categories is underway. This study exemplified the potential application of CNFB in studying cell differentiation, provided useful insights for manipulating in vitro hair lineage differentiation of epithelial cells, facilitating future design of bioengineering products.