Our laboratory has reported that somatotroph differentiation occurs between day 14 and day 16 of chicken embryonic development and that corticosterone (CORT) can induce somatotroph differentiation at an earlier age in vitro and in vivo. The objective of the present study was to test for thyroid hormone-CORT interactions on somatotroph differentiation in vitro. Pituitary cells from day 11 chicken embryos were treated with CORT and thyroid hormones, and growth hormone (GH) producing somatotrophs were detected by reverse hemolytic plaque assays and immunocytochemistry. We found that thyroid hormones can act synergistically with CORT to further augment the abundance of somatotrophs in vitro, while having little to no effect on their own. Both thyroxine (T₄) and triiodothyronine (T₃) could act synergistically with CORT to increase somatotroph abundance, but the effects of T₃ were biphasic, inhibiting CORT actions at higher concentrations. The monodeiodination inhibitor iopanoic acid (IOP) inhibited the synergistic effect of T₄ on CORT induction of GH cells in vitro, but not the synergistic effect of CORT and T₃ or the effect of CORT alone. Furthermore, T₃ treatment overcame the IOP-induced reduction in the T₄-CORT effect. Our findings indicate that thyroid hormones act synergistically with CORT to further augment the abundance of somatotrophs in vitro and that conversion of T₄ to T₃ within the pituitary is involved in T₄ modulation of somatotroph abundance. Somatotroph differentiation during normal development may be regulated by complex interactions of hormones produced by the embryonic thyroid and adrenal glands.