Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts. Display omitted •EPS8 and IRTKS puncta mark sites of new microvillus growth•EPS8 and IRTKS puncta remain enriched at the distal tips of nearly all microvilli•Existing microvilli also serve as mothers that give rise to daughter protrusions•Microvilli collapse when membrane wrapping and EPS8/IRTKS tip enrichment are lost Gaeta et al. show that microvillus biogenesis occurs de novo and from existing microvilli, with the actin binding proteins EPS8 and IRTKS marking new sites of growth. Nascent microvilli also undergo collapse, whereby the protrusion initially loses membrane wrapping and EPS8/IRTKS tip enrichment, ultimately leading to loss of the core actin bundle.