We have constructed a recombinant fowlpox virus expressing HIV antigens and the costimulatory molecule 4-1BBL. When included in the boost, but not the prime of a poxvirus prime-boost strategy, 4-1BBL significantly enhanced the anti-HIV T cell response generated to this vaccination in BALB/c mice, as detected by ex vivo IFNγ ELISPOT responses, intracellular cytokine staining to HIV Gag antigens, and enumeration of Gag-reactive CD8 T cells. 4-1BBL however, is not capable of modulating the CD4 T cell response, nor the antibody response to this vaccination strategy. Enhancement of the T cell response by 4-1BBL continues into the memory phase, as detected 2 months post vaccination. This data is the first to show modulation of the immune response to a viral vaccine by coexpression of 4-1BBL and supports this strategy as an exciting approach for enhancement of T cell memory in prime-boost vaccines.