Aim Prenatal hypoxia due to placental insufficiency results in deleterious phenotypes and compensatory mechanisms including increased sympathetic tone. Utilizing the embryonic chicken model, we investigated (i) changes in nitric oxide (NO)‐mediated tone in response to chronic hypoxic development and (ii) the in vivo role of NO‐mediated tone during acute hypoxic exposure, which has not been previously studied. We hypothesized that NO tone on the cardiovascular system would be unaffected by chronic hypoxic incubation in White Leghorn chicken (Gallus domesticus) embryos. Methods We measured arterial pressure, heart rate and femoral blood flow (via a Doppler flow probe) in response to acute hypoxia (10% O2) and pharmacological manipulations in normoxic‐ and hypoxic (15% O2)‐incubated embryos. This was performed at 70 and 90% of total incubation time (21 days). At 70% of incubation (day 15), blood volume and chorioallantoic membrane development are maximal; 90% of incubation (day 19) is 1 day prior to lung ventilation. Results Acute hypoxic exposure decreased femoral flow in both 90% groups, but increased femoral artery resistance in the hypoxic group. NO tone increased during development, but was not affected by hypoxic incubation. Inhibition of NO production by L‐NAME (100 mg kg−1) revealed that NO plays a significant role in the flow response to hypoxia. Conclusion Chronic hypoxic incubation has no effect on cardiovascular NO tone during White Leghorn chicken development. In the intact animal, NO function during acute hypoxic stress is suppressed by hypoxic incubation, indicating that chronic hypoxic stress dampens the NO contribution.