T-bet and GATA-3 are known to regulate cytokine expression in T lymphocytes, and cytokines have been implicated in endometrial regulation and implantation. Previous work showed that female steroid hormones modulate the expression of T-bet in endometrial epithelial cells, suggesting a mechanism for local immune regulation in the human endometrium. We hypothesized that stromal cells are involved in immune regulation, as they have been shown to exert paracrine effects on other endometrial cells and compartments and also secrete cytokines. The objective of this study was to examine the modulation of the gene expression of T-bet and GATA-3, and of the cytokines interferon γ (IFN-γ) and interleukin 4 (IL-4), by female steroid hormones, in human endometrial stromal cells (HESC) in long-term cultures (30 days) mimicking the normal menstrual cycle. T-bet and GATA-3 messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction, and intracellular protein production was demonstrated by immunoblotting. In addition, secretion of IL-4 and IL-15 was measured by enzyme-linked immunosorbent assay. T-bet and IL-4 mRNA expression increased and GATA-3 decreased under decidualization conditions; IFN-γ was not detected. Secretion of IL-15 increased during decidualization, and IL-15 upregulated T-bet gene expression. In conclusion, gene expression of T-bet and GATA-3 by endometrial stromal cells is under hormonal conditions mimicking decidualization, and the results are consistent with an autocrine regulatory mechanism of IL-15 secretion and T-bet expression.