The positive selection of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated Ag presentation by CD4+CD8+thymocytes. Maturation of newly selected iNKT cells continues in the periphery and also involves CD1d expression. CD1d molecules acquire Ags for presentation in endosomal compartments, to which CD1d molecules have access through an intrinsic CD1d-encoded tyrosine motif and by association with the class II MHC chaperone, invariant chain. In this study, we report the generation of mice in which all CD1d is replaced by CD1d-enhanced yellow fluorescent fusion protein (EYFP). CD1d-EYFP molecules are stable, present lipid Ags, and have near normal subcellular distribution. CD1d-EYFP molecules mediated positive selection of Vα14 iNKT cell precursors at decreased efficiency, caused a delay in their terminal maturation, and did not invoke Vα14 iNKT cell effector function as wild-type CD1d could. Using these mice, we show that the intrinsic CD1d-encoded sorting motif mediates thymic selection and activation of Vα14 iNKT cells by professional APCs, while for peripheral terminal differentiation the intrinsic CD1d sorting motif is dispensable. Copyright