Short-chain fatty acids (SCFAs; butyrate and propionate) up-regulate embryonic/fetal globin gene expression through unclear mechanisms. In a murine model of definitive erythropoiesis, SCFAs increased embryonic beta-type globin gene expression in primary erythroid fetal liver cells (eFLCs) after 72 hours in culture, from 1.7% (+/- 1.2%) of total beta-globin gene expression at day 0 to 4.9% (+/- 2.2%) in propionate and 5.4% (+/- 3.4%) in butyrate; this effect was greater in butyrate plus insulin/erythropoietin (BIE), at 19.5% (+/- 8.3%) compared with 0.1% (+/- 0.1%) in ins/EPO alone (P < .05). Fetal gamma-globin gene expression was increased in human transgene-containing eFLCs, to 35.9% (+/- 7.0%) in BIE compared with 4.4% (+/- 4.2%) in ins/EPO only (P < .05). Embryonic globin gene expression was detectable in 11 of 15 single eFLCs treated with BIE, but in0 of 15 ins/EPO-only treated cells. Butyrate-treated [65.5% (+/- 9.9%)] and 77.5% (+/- 4.0%) propionate-treated eFLCs were highly differentiated in culture, compared with 21.5% (+/- 3.5%) in ins/EPO (P < .005). Importantly, signaling intermediaries, previously implicated in induced embryonic/fetal globin gene expression (STAT5, p42/44, and p38), were not differentially activated by SCFAs in eFLCs; but increased bulk histone (H3) acetylation was seen in SCFA-treated eFLCs. SCFAs induce embryonic globin gene expression in eFLCS, which are a useful short-term and physiologic primary cell model of embryonic/fetal globin gene induction during definitive erythropoiesis.