Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-gamma, interferon-alpha, tumor necrosis factor-alpha, Poly I:C, and IL1-beta (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-gamma alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.