AbstractPax9 is required for the formation of a variety of organs during mouse development. The function of Pax9 at postnatal stages is unknown since homozygosity of the null allele (Pax9lacZ) causes neonatal lethality. Recently, we have generated a hypomorphic Pax9 allele, Pax9neo, which contains a removable neomycin resistance cassette (neo) and loxP sites flanking the first two exons of Pax9. Here we show that FLP‐mediated in vivo excision of neo generates phenotypically normal Pax9flox mice. Crossing Pax9flox mice to PGK‐Cre mice leads to efficient recombination of loxP sites and neonatal lethality in the resulting Pax9del/del offspring. Inactivation of Pax9 using Wnt1‐Cre mice causes cleft secondary palate and tooth agenesis and reveals that the Pax9 expressing mesenchymal cells of the nose, palate, and teeth are derived from neural crest cells. The conditional Pax9 allele will be a valuable tool to study Pax9 function in specific tissues of adult mice. genesis 45:460–464, 2007. © 2007 Wiley‐Liss, Inc.