Retinal progenitor cells (RPCs) undergo a series of changes over time that affect their competency to produce different cell types at different times in development. The transcriptional machinery that regulates these changes, as well as associated gene expression changes, have not been characterized. An analysis of the regulatory region of the retinal homeodomain transcription factor, Chx10, was carried out using in ovo electroporations in chick and transgenic mice. An RPC enhancer was defined that mediates reporter activity in subsets of RPCs and directs high-level expression in intermediate and late RPCs. Using bioinformatic and biochemical analysis, a key binding site in this enhancer was found and was shown to be bound by the POU domain factors, Brn-2 and Tst-1/SCIP, in retinal extracts. Analysis of the Brn-2 expression pattern shows that it is expressed in intermediate and late RPCs, but not early RPCs, and thus partially overlaps with expression of the reporter activated by the defined Chx10 enhancer. Biochemical analysis also revealed binding of both Chx10 and Brn-2 to an enhancer of the CNS progenitor cell marker, Nestin. Nestin expression in the retina is restricted to intermediate/late RPC subsets, and genetic evidence is presented that demonstrates that Chx10 represses Nestin expression in early RPCs. A bipolar cell enhancer for Chx10 also was defined, and a role for Brn-2 in expression of Chx10 in bipolar cells is predicted. These data identify Brn-2 as a new marker of subsets of RPCs and suggest a mechanism by which a combination of POU factors and Chx10 define RPC gene expression patterns, such as that of Nestin.