Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual Id (inhibitor of differentiation) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared to levels of Id1, Id3 and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3 and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, as factors that promote inhibitory MAPK signaling (transforming growth factor and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3 and Id4. Furthermore, over-expression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3 and Id4 mRNA, but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knock-down studies using siRNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA, and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.