Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain the highly specialised erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced band 3 and protein 4.2 expression. We use in vitro differentiation of erythroblasts transduced with shRNAs targeting the ANK1 gene to generate erythroblasts and reticulocytes with a novel ankyrin-R ‘near null’ human phenotype with less than 5% of normal ankyrin expression. Using this model we demonstrate that absence of ankyrin negatively impacts the reticulocyte expression of a variety of proteins including band 3, glycophorin A, spectrin, adducin and more strikingly protein 4.2, CD44, CD47 and Rh/RhAG. Loss of band 3, which fails to form tetrameric complexes in the absence of ankyrin, alongside GPA, occurs due to reduced retention within the reticulocyte membrane during erythroblast enucleation. However, loss of RhAG is temporally and mechanistically distinct, occurring predominantly as a result of instability at the plasma membrane and lysosomal degradation prior to enucleation. Loss of Rh/RhAG was identified as common to erythrocytes with naturally occurring ankyrin deficiency and demonstrated to occur prior to enucleation in cultures of erythroblasts from a hereditary spherocytosis patient with severe ankyrin deficiency but not in those exhibiting milder reductions in expression. The identification of prominently reduced surface expression of Rh/RhAG in combination with direct evaluation of ankyrin expression using flow cytometry provides an efficient and rapid approach for the categorisation of hereditary spherocytosis arising from ankyrin deficiency.