We have used an in vitro system to study the effects of major histocompatibility complex class I binding peptides on thymic development. Fetal thymus lobes from mice deficient in the class I light chain (β2 microglobulin or β2-/-) were cultured for 10 d in vitro, during which time T cell precursors develop into mature T cells. In these organ cultures, as in the adult or neonatal β2M-/- thymus, CD8+ mature T cells did not develop, demonstrating that the mature T cells seen during early murine thymic development are the result of the positive selection process. To these cultures we added various class I binding peptides with or without a source of exogenous β2M. CD8+ T cells developed to various degrees only in the presence of β2M and peptides. Using peptide mixtures of differing complexity, we showed that the efficiency of this process is dependent more on peptide complexity than on peptide concentration. These data argue for a specific role for peptides in the process of positive selection. Furthermore, this culture system should be useful in identifying peptides that can promote positive selection of cells expressing a specific T cell receptor (TCR) in TCR transgenic mice.