Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.