The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the growth hormone (GH) producing somatotropes and the prolactin (PRL) producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors, that contribute to the somatotrope and lactotrope cell identities. Here, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of mRNAs that are selectively enriched in each of the two populations includes a set of transcription factors. A subset of these factors may have roles in pituitary lineage divergence, and/or in regulating expression of cell-type specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope-enriched and two lactotrope-enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark somatotrope or lactotrope genes in a pituitary-derived cell culture model system. Conditional inactivation of the genes encoding these factors in mice revealed that two of these transcription factors impact the expression of landmark hormone genes and alter cell phenotypes in primary murine pituitary. Subsequent mechanistic studies revealed that these two factors act on their target genes either through a direct binding at the target promoter and release of paused Pol II complexes, or through indirect mechanism(s). The functions of these two factors are further remarkable in how they maintain hormone expression in the lactotrope and somatotrope lineages; one serves to enhance landmark Prl gene expression in lactotropes while the second serves to reciprocally repress expression of the Prl gene in somatotropes. In conclusion, these studies identify novel regulators of the somatotropes and lactotropes, explore their mechanisms of action, and establish a useful database for further study of these lineages in the anterior pituitary.