The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+ T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28(-/-)) yielded normal levels of antigen- specific interferon γ production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishmania major, C57BL6 CD28(-/-) mice were fully capable of controlling infection and exhibited a normal T helper T response. BALB/c CD28(-/-) mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28(-/-) mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28(-/-) mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4-producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.