The pineal complex of zebrafish consists of a pineal organ and a left-sided parapineal organ. Mutation of the floating head (flh) gene, which encodes a homeodomain protein, causes premature termination of pineal cell division without affecting specification or asymmetric placement of the parapineal. The from beyond (fby) mutation, a premature stop codon in the T-domain-containing protein Tbx2b, disrupts formation of the parapineal while leaving the pineal largely intact. However, flh is reported as being required for tbx2b transcription. To resolve the paradox that flhand tbx2b mutants have opposite phenotypes but have been placed in the same genetic pathway, we have examined transcriptional cross-regulation in single flh or fby mutants and genetic epistasis in double mutants. Careful analysis shows that flh is not required for tbx2b transcription and double mutants exhibit an additive phenotype. We conclude that Flh and Tbx2b regulate separate programs of pineal and parapineal development. Developmental Dynamics 237:3538–3544, 2008. © 2008 Wiley-Liss, Inc.