Segmentation of the vertebrate hindbrain into multiple rhombomeres is essential for proper formation of the cerebellum, cranial nerves and cranial neural crest. Paralog group 1 (PG1) hox genes are expressed early in the caudal hindbrain and are required for rhombomere formation. Accordingly, loss of PG1 hox function disrupts development of caudal rhombomeres in model organisms and causes brainstem defects, associated with cognitive impairment, in humans. In spite of this important role for PG1 hox genes, transcriptional targets of PG1 proteins are not well characterized. Here we use ectopic expression together with embryonic dissection to identify novel targets of the zebrafish PG1 gene hoxb1b. Of 100 genes up-regulated by hoxb1b, 54 were examined and 25 were found to represent novel hoxb1b regulated hindbrain genes. The ppp1r14al gene was analyzed in greater detail and our results indicate that Hoxb1b is likely to directly regulate ppp1r14al expression in rhombomere 4. Furthermore, ppp1r14al is essential for establishment of the earliest hindbrain signaling-center in rhombomere 4 by regulating expression of fgf3.