In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.