Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ specific Hb thresholds for tissue hypoxia, HIF-α (ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50g/L, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and -2α increased earlier (Hb~70-90g/L) in response to anemia. Liver also exhibited an early HIF-1α response. Carotid blood flow was increased early (Hb~70g/L) but renal blood flow remained relatively constant, only increased at Hb of 50g/L. Anemia increased nNOS (brain and kidney) and eNOS (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS-independent. HIF-dependent RNA levels increased linearly (~10 fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (~100 fold). Plasma EPO levels increased near Hb threshold of 90g/L, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.