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Much of the current understanding of the β → α + β phase transformation in the commercially important titanium alloy Ti-6Al-4V (Ti64) is by inference from more β-stabilised analogues, or post-mortem studies. In-situ cooling experiments have been conducted for the first time on Ti64 samples, in an adapted SEM with a high temperature heating stage, while performing real-time secondary electron video recording and sequential electron backscatter diffraction mapping. This has enabled direct observation of the development of grain boundary (GB) α colony microstructures, when cooling at relatively low rates (0.3 – 0.1°C s−1) through the β transus. It is shown that the α colonies develop discontinuously, with relatively widely spaced larger ‘primary’ α lamellae nucleating first at lower undercoolings at β GBs, which grow out rapidly into the grains until impingement. These primary laths form a ‘skeletal template’ for the subsequent colonies by determining the variant selected within their domain of influence, as the extent of transformation increases. This occurs through biasing the subsequent sympathetic autocatalytic nucleation of finer secondary α laths combined with branching and plate broadening, to ‘infill’ the remaining β matrix with laths of the same variant. Evidence has also been provided that is in agreement with previous studies of the crystallographic relationships between the nucleating GB α allotriomorph variants, colonies, and the β GBs. However, GB α allotriomorphs were not always found to be a pre-requirement for the development of single variant α colonies in Ti64 (at least when observed at a surface), with the primary α laths seen to nucleate directly off β GBs in competition with the GB α allotriomorphs in many cases.