AbstractThe Runx (runt‐related) family of transcription factors are important regulators of cell fate decisions in early embryonic development, and in differentiation of tissues including blood, neurons, and bone. During skeletal development in mammals, while only Runx2 is essential for osteoblast differentiation, all family members seem to be involved in chondrogenesis. Runx2 and Runx3 control chondrocyte maturation. Both Runx1 and Runx2 are expressed early in mesenchymal condensations, but how they contribute to the initial stages of chondrocyte differentiation is unclear. Here we show that a hierarchy of Runx transcriptional regulation promotes the early program of chondrocyte differentiation from pre‐cartilage mesenchyme in the zebrafish head skeleton. We have previously characterized the zebrafish orthologs for all Runx genes. Zebrafish runx2 is duplicated, but not runx1 or runx3. In the work presented here, we determined the early expression pattern of the runx genes in the craniofacial region. The earliest expression detected was that of runx3 in the pharyngeal endoderm, then runx2a and b in mesenchymal condensations, and later runx1 in the epithelium. Using antisense morpholino knockdown analysis, we examined their respective activities in early chondrogenesis. Depletion of runx2b (but not runx2a) and runx3 severely compromised craniofacial cartilage formation. Because runx2b expression was abolished in Runx3 morphants, we propose that endodermal Runx3 has a role in influencing signaling activities from the endoderm to promote chondrocyte differentiation. We also show that, in contrast to data from mouse studies, zebrafish Runx1 is not required in the initial steps of chondrogenesis leading to endochondral bone formation. Developmental Dynamics 235:3166–3176, 2006. © 2006 Wiley‐Liss, Inc.