To investigate the breeding and genotyping for Tbx18 knock-out mice (Tbx18:Cre knock-in Mus musculus) and the application of Tbx18 knock-out and genetic tracing mice, we introduced into Tbx18:Cre knock-in heterozygote mice and breed offspring. The genotyping of the offspring were performed by PCR using genomic DNA. We observed the morphology of knock-out (KO) hearts from male and female heterozygate mice inter-crossed by Hematoxylin and eosin-staining(H.E)analysis. We also observed the fate of Tbx18-expressing epicardial cells within Tbx18:Cre/Rosa26REYFP embryos by cryostat sections. The results indicated that the genotyping of heterozygote offspring, Tbx18 knock-out and Tbx18:Cre/Rosa26REYFP embryos were consistent with Mendel's law of segregation. We also found that there was malformation of the SAN head in Tbx18 knock-out heart compared with the wild-type. The Tbx18-expressing epicardial cells were important cardiac progenitor resource in mouse heart development. These results suggest that Tbx18 knock-out mice is an ideal model for studying the mechanisms of congenital heart diseases. The Tbx18-expressing epicardial cells are candidate cardiac progenitor for cardiac repair and regeneration in heart disease.