Erythroid terminal differentiation is the process where red blood cells undergo significant morphological and functional changes prior to entering the blood stream. Characteristic of this is erythroid enucleation, a feature unique to mammalian erythroblasts that has been established as the rate limiting factor for the ex vivo synthesis of red blood cells. The enucleation event is a distinctive cell division, where the enucleated reticulocyte enters the blood stream and undergoes further maturation into a fully functioning erythrocyte, and the pyrenocyte containing the expelled nuclei, is degraded by macrophages in the bone-marrow microenvironment. Cyclin-dependent kinase-9 (CDK9) has been identified a novel regulator of enucleation, however the precise function and expression of CDK9 in human erythroid terminal differentiation is currently unknown. Here, I have established a number of validations and novel findings, including characterisation of the Human umbilical cord-blood derived erythroid progenitor-2 (HUDEP-2) cell line as a model for erythroid enucleation, the expression and localisation of CDK9 in human erythroid differentiation, as well as establishing a genetic system to identify the function of CDK9 in this process. The HUDEP-2 system effectively generates orthochromatic erythroblasts; however, the rate of enucleation is low, with many cells arresting in the final stages. CDK9 is expressed and localised to both the cytoplasm and nucleus of differentiating HUDEP-2 cells. Additionally, CDK9 appears to be strongly expressed in the reticulocyte body of enucleating cells. The functionality of CDK9 in human erythropoiesis remains unknown, however generation of a CDK9 analogue-sensitive HUDEP-2 stable cell line by CRISPR-Cas9 technology will allow future studies to elucidate the function of CDK9 in erythropoiesis.