160 words) 1 Centrosome-mediated microtubule (MT) nucleation has been well-characterized; however, 2 numerous non-centrosomal MT nucleation mechanisms exist. The branching MT nucleation 3 pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin 4 complex to initiate nucleation of new MTs. While the pathway is well-conserved at a molecular 5 and functional level, branching MT nucleation by core constituents has never been directly 6 observed in animal cells. Here, multi-color TIRF microscopy was applied to visualize and 7 quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells. 8 The stereotypical branching nucleation event entailed augmin first binding to a "mother" MT, 9 recruitment of γ-TuRC after 16s, followed by nucleation 15s later of a "daughter" MT at a 36° 10 branch angle. Daughters typically remained attached throughout their ~40s lifetime unless the 11 mother depolymerized past the branch point. Assembly of branched MT arrays, which did not 12 require D-TPX2 (Drosophila TPX2) or evident regulation by a RanGTP gradient, enhanced 13 localized RhoA activation during cytokinesis. 14 15