Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10^Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10^Ki–v1, to target FGF10^Pos cells. While this line allowed fairly efficient and specific labeling of FGF10^Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10^Cre–ERT2 line (called thereafter Fgf10^Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10^Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10^Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10^Ki–v2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10^Ki–v2 mouse is capable of significantly targeting FGF10^Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10^Ki–v2 line opens the way for future mechanistic experiments involving the postnatal lung.