Differential tumour cell behaviour caused by environmental conditions, termed dynamic heterogeneity, is a prime source for drug resistance. We utilize real-time cell cycle imaging (FUCCI) to study melanoma heterogeneity in vitro and in vivo. As distinct proliferative and invasive capabilities reflect differential drug sensitivities, identifying and characterising these different responses is crucial to design effective therapies. Mouse xenograft tumours generated from cell lines with high microphthalmia-associated transcription factor (MITF) level displayed a homogeneous distribution of cycling cells throughout. In contrast, tumours generated from cell lines with low MITF levels were composed of clusters of cycling cells and clusters of G1-arrested cells. The proliferating areas were in close proximity to blood vessels, presumably characterised by oxygen/nutrient availability. Melanoma spheroids recapitulated the in vivo cycling behaviour, considering that here oxygen and nutrients are supplied by diffusion. MITF and its transcriptional targets were undetectable within the hypoxic G1-arrested spheroid core, indicating hypoxia-induced MITF downregulation. Modulation of MITF expression impacted spheroid morphology, with overexpression giving rise to larger but flatter structures whereas knock-down resulted in smaller aggregates with unaffected sphericity. Surprisingly, the loss of morphological integrity caused by increased MITF expression did not reduce the spheroids’ inner hypoxic level, dismissing the hypothesis that these compromised structures could be be more permeable to oxygen and nutrients resulting in G1-arrest induced by decreased hypoxia/starvation. We therefore conclude that MITF protects from cell cycle arrest induced by oxygen/nutrient deprivation. We hypothesize that high MITF levels prevent cell cycle arrest by reducing the cell-intrinsic propensity to arrest in response to low oxygen/nutrient and we are currently undertaking proteomics studies to elucidate the underlying molecular mechanisms. Taken together, MITF is a potent regulator of dynamic heterogeneity, which in turn impacts on drug sensitivity.