The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines, which act through receptor complexes containing the signal transducer gp130. The study investigates the downstream mechanisms leading to ES cell self-renewal. Chimeric receptors were introduced into ES cells, and biochemical and functional studies demonstrated the requirement for engagement and activation of the latent transcription factor STAT3. Detailed mutational analyses revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was further investigated using the dominant interfering mutant, STAT3F. ES cells expressing this molecule could not be isolated, but an episomal supertransfection strategy enabled the consequences of STAT3F expression to be examined. Additionally, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These findings establish that STAT3 plays a central role in maintaining the pluripotential stem cell phenotype, contrasting its involvement in somatic cell differentiation. The identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a significant step in understanding the molecular characterization of pluripotency.The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines, which act through receptor complexes containing the signal transducer gp130. The study investigates the downstream mechanisms leading to ES cell self-renewal. Chimeric receptors were introduced into ES cells, and biochemical and functional studies demonstrated the requirement for engagement and activation of the latent transcription factor STAT3. Detailed mutational analyses revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was further investigated using the dominant interfering mutant, STAT3F. ES cells expressing this molecule could not be isolated, but an episomal supertransfection strategy enabled the consequences of STAT3F expression to be examined. Additionally, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These findings establish that STAT3 plays a central role in maintaining the pluripotential stem cell phenotype, contrasting its involvement in somatic cell differentiation. The identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a significant step in understanding the molecular characterization of pluripotency.