Topical application of antigen induces antigen‐specific humoral and cellular immune responses. In this study we examined whether expansion of dendritic cells (DC) by Flt3 ligand (Flt3L) treatment influences the induction of immune responses following transcutaneous immunization. Mice were treated intraperitoneally with Flt3L or phosphate‐buffered saline (PBS) and immunized transcutaneously with hen egg lysozyme (HEL). Flt3L‐treated mice developed lower HEL‐specific cellular and humoral immune responses than PBS‐treated mice. However, in the presence of cholera toxin (CT), a potent adjuvant for mucosal and transcutaneous immunization, Flt3L‐treated mice developed significantly higher cellular and humoral immune responses to HEL when compared to PBS‐treated mice. We assessed whether the immunomodulatory effects of CT were a result of activation of epidermal dendritic cells (Langerhans' cells; LC). Our results indicate that within 8–12 hr of topical application of CT, epidermal LC cells lose their dendritic morphology and become rounder in appearance. In addition, we observed enhanced expression of major histocompatibility complex (MHC) class II, and of adhesion molecules CD11c and intracellular adhesion molecule‐1 (ICAM‐1). Our observations support the concept that the state of activation of DC in the skin is central to the regulation of immune responses. This information is relevant to the design of effective transcutaneous vaccination strategies.