Although much progress has been made in understanding the role of NK cells in bone marrow transplantation, little is known about their function in CD4 T cell-mediated allograft rejection. We have previously shown that in the absence of CD8 T lymphocyte priming, the in vivo default development pathway of alloreactive CD4 T cells was strongly biased toward Th2 phenotype acquisition. In this study, we investigate the impact of NK cells on the activation and differentiation of alloreactive CD4 T cells in various donor/recipient combinations. Our data demonstrate that defective inhibition of host NK cells by donor APCs including dendritic cells (DCs) results in diminished allospecific Th cell responses associated with the development of effector Th cells producing IFN-gamma rather than type 2 cytokines. Turning host NK cells off was sufficient to restore strong alloreactive CD4 T cell priming and Th2 cell development. Similar results were obtained by analyzing the effect of NK cell activation on CD4 T cell responses to skin allografts. However, despite the dramatic effect of NK cells on alloreactive Th1/Th2 cell development, the kinetics of skin graft rejection were not affected. Thus, Th2 differentiation is a major pathway of alloreactive CD4 T cell development during solid organ transplant rejection, as long as host NK and CD8 T cells are not activated. We propose the hypothesis that MHC class I-driven interactions between donor DCs and host NK cells or CD8 T cells might result in DC-carried signals controlling the dynamics of alloreactive CD4 T cell priming and polarization.