Lens development is regulated by a variety of transcription factors with distinct properties. The lens-specific transcription factor, L-Maf, is essential for lens formation and induces lens-specific markers, such as the crystallin genes. In this study, we analyzed the mechanism by which L-Maf regulates delta-crystallin expression. Misexpression of L-Maf in the head ectoderm of lens placode-forming embryos by in ovo electroporation induced delta-crystallin only in the region surrounding the lens. To define this restricted expression, we misexpressed L-Maf together with other transcription factors implicated in delta-crystallin expression. Sox2 plus L-Maf expanded the delta-crystallin-inducible domain to the entire head ectoderm and simultaneously increased the quantity of delta-crystallin mRNA expressed. In contrast, co-expression of L-Maf with other factors such as Pax6, Six3 and Prox1 had little or no effect on delta-crystallin. We also observed that L-Maf and Sox2 cooperatively enhanced the transactivation of a reporter gene bearing the delta-crystallin enhancer in ovo, implying that L-Maf and Sox2 can induce delta-crystallin through the same enhancer. In conclusion, we report here that L-Maf and Sox2 cooperatively regulate the expression of delta-crystallin during chick lens development.