Triiodothyronine (T3) is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonal carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3-target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CamKIV), in response to T3. Full T3-induction in transient transfection of EC cells was restored with co-transfection of a TR-expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem cells compared with expression in cells with deficient (EC) or mutant TR (TR P398H mutant ES cells), to identify T3-target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only about 60 genes (1%) met criteria for direct T3-stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3-target genes; neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3-responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.