The transcription factors RUNX2 and OSX have been shown to act sequentially to direct mammalian osteoblast differentiation. RUNX2 is required during the early stages of commitment and acts in part to activate Osx transcription. OSX and RUNX2 then act to direct transcription of bone matrix proteins. Here, we investigate the expression of these genes and others during zebrafish osteoblastogenesis. Using whole-mount in situ hybridization, we find that, during the formation of a given bone, the zebrafish homologues of mouse Runx2 (runx2a and runx2b) are typically expressed before the onset of osx. osx expression is usually followed by up-regulation of the bone matrix proteins, col1a2 and osteonectin. These results suggest that the mammalian pathway is conserved during development of the head and shoulder skeleton of zebrafish. We also analyze the expression of three atypical bone markers (tcf7, cvl2, and col10a1) in an effort to place them within this canonical hierarchy.