TG-interacting factor (TGIF) is a TALE homeodomain protein expressed predominantly in the central nervous system and functions as a transcriptional repressor. Several mutations in TGIF have been identified in patients with holoprosencephaly, the most common congenital malformation of the developing human forebrain. However, the precise role of TGIF in neural development is not well understood. We found that TGIF was expressed strongly in the mouse retina during early stages of development, and that its expression gradually decreased as retinal development progressed. In vitro explant cultures of mouse retina mimic the in vivo development of retinal subtypes. Forced expression of TGIF using a retrovirus in explant culture induced the differentiation of amacrine cells from retinal progenitor cells. A TGIF paralog, TGIF2, showed a similar transition in expression during retinal development, and TGIF2 also promoted amacrine cell differentiation in a retinal explant culture system. However, no apparent difference between wild-type and TGIF-knockout mouse retina was observed, suggesting that TGIF and TGIF2 function redundantly in that tissue. Forced expression of TGIF homeodomain (HD)-EnR (repressing) rather than TGIF HD-VP16 (activating) resulted in a phenotype similar to that induced by wild-type TGIF, suggesting that TGIFs may act as transcriptional repressors to induce amacrine genesis.