Recent advances in our understanding of iron metabolism regulation and crosstalk with erythropoiesis have provided insight into the pathophysiology of multiple disease conditions. For instance, the peptide hormone hepcidin is central to the regulation of iron metabolism. Its effect on cellular iron concentration involves binding ferroportin, the main iron export protein, resulting in its internalization and degradation and leading to iron sequestration within ferroportin-expressing cells. Furthermore, hepcidin regulation by erythropoiesis is attributed in large part to a bone marrow–derived hormone erythroferrone. Erythroferrone-induced hepcidin suppression in diseases of expanded hematopoiesis results in iron overload. Conversely, diseases, such as iron refractory iron deficiency anemia and anemia of chronic inflammation, are characterized by aberrantly increased hepcidin, resulting in iron sequestration and decreased circulating iron and eventually leading to iron-restricted erythropoiesis. Lastly, because iron functions in concert with erythropoietin to promote erythroid precursor survival, proliferation, and differentiation, iron deficiency anemia is a consequence not only of decreased hemoglobin synthesis in each cell but also, a decrease in erythropoietin responsiveness in the bone marrow. How to translate this new information to the clinical setting has not been fully elucidated. The purpose of this manuscript is to summarize current standard tools for identifying iron deficiency in anemic patients; explore the tools and context for evaluating novel markers, such as hepcidin, erythroferrone, and markers of the iron restriction response; and assess available evidence for how their use could increase our understanding of health outcomes in clinically challenging cases.