Abstract The TNF superfamily members CD70 and OX40 ligand (OX40L) were reported to be important for CD4+ T cell expansion and differentiation. However, the relative contribution of these costimulatory signals in driving CD4+ T cell responses has not been addressed. In this study, we found that OX40L is a more important determinant than CD70 of the primary CD4+ T cell response to multiple immunization regimens. Despite the ability of a combined TLR and CD40 agonist (TLR/CD40) stimulus to provoke appreciable expression of CD70 and OX40L on CD8+ dendritic cells, resulting CD4+ T cell responses were substantially reduced by Ab blockade of OX40L and, to a lesser degree, CD70. In contrast, the CD8+ T cell responses to combined TLR/CD40 immunization were exclusively dependent on CD70. These requirements for CD4+ and CD8+ T cell activation were not limited to the use of combined TLR/CD40 immunization, because vaccinia virus challenge elicited primarily OX40L-dependent CD4 responses and exclusively CD70-dependent CD8+ T cell responses. Attenuation of CD4+ T cell priming induced by OX40L blockade was independent of signaling through the IL-12R, but it was reduced further by coblockade of CD70. Thus, costimulation by CD70 or OX40L seems to be necessary for primary CD4+ T cell responses to multiple forms of immunization, and each may make independent contributions to CD4+ T cell priming.