Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope±cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1α plus IL-18, IL-1α plus IL-12, and IL-1α plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60–71% peptide-specific 51 Cr release). Peak H-2D d-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8+ PBMC. Nasal immunization with HIV immunogen and IL-1α, IL-12, and GM-CSF also induced Ag-specific IFN-γ-secreting cells in the draining cervical lymph node and the lung. The use of IL-1α, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7. 1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1α, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.