The hygiene hypothesis implies that the increasing prevalence of allergy in ‘westernized’ countries is explained by reduced bacterial exposure in early life, but the underlying mechanism remains elusive. We therefore wanted to study the effect of bacterial lipopolysaccharide (LPS) on the generation of regulatory T (TR) cells in neonates, and to analyze differences between neonates with allergy risk because of a family history of atopy (FH+) and controls without such hereditary risk (FH−). Cord blood mononuclear cells from the FH+ and FH− groups were stimulated with β-lactoglobulin in the presence of LPS. T-cell phenotypes suggestive of TR cells [CD25+, CD25high and integrin (CD103+)], and the intracellular proliferation antigen Ki-67 were quantified by flow cytometry. Release of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) from its inactive complex was determined by enzyme-linked immunosorbent assay. The analyses revealed the generation of T-cell phenotypes suggestive of TR cells including a CD25high T-cell subset which was inversely related to T-cell proliferation (r = −0.54, p < 0.05) and to activation-induced release of TGF-β1 (r = −0.80, p < 0.001). The CD25high T-cell subset tended to be impaired in the FH+ group (% of CD3+ T cells: FH+, 5.1% vs. FH−, 12.6%), and notably, the FH+ group showed a significantly reduced capacity for generation of both CD25+ (FH+, 16.2% vs. FH−, 34.9%; p < 0.01) and T cells (FH+, 2.1% vs. FH−, 3.9%; p < 0.05). Our findings suggested that early-life exposure to a dietary antigen in the presence of LPS might modulate the immune system by generating TR cells. This capacity was impaired in neonates with hereditary allergy risk, but clinical follow-up will be required to determine a possible effect on allergy emergence.