AbstractLaminin‐332 pemphigoid is a rare and chronic autoimmune blistering disease which results in subepidermal blisters and erosive lesions predominantly localized to mucous membranes. As histologic inflammation is variable and non‐complement‐fixing IgG antibodies against laminin‐332 are the predominant class of autoantibodies deposited at the epidermal basement membrane zone, we hypothesized that complement‐independent pro‐inflammatory and blistering pathways existed similarly to that previously shown in bullous pemphigoid. As autoantibodies to laminin α3 are most prevalent, we studied the major cellular response to blockade of laminin α3 using a well‐characterized monoclonal antibody (P3H9‐2). RNA‐seq revealed upregulation of numerous desmosomal genes (DSG1, DSG3, DSC1, DSC3 and DSP) as well as KRT1 and KRT10. Additionally, P3H9‐2‐treated cells demonstrated downregulation of most hemidesmosomal genes. A pro‐inflammatory response was not appreciated. Using pharmacological inhibitors, we identified both protein kinase C and NOTCH as key regulators of P3H9‐2 induced differentiation. We lastly utilized 3D human skin equivalents to determine whether blockade of laminin α3 would lead to delayed blistering, consistent with keratinocyte differentiation. Significant blistering was noted after 72 h of treatment, with only minimal separation at 24 h. In summary, blockade of laminin α3 alters keratinocyte differentiation, representing a potential complement‐independent mechanism of blistering.