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Summary: The implementation of in vitro approaches using undifferentiated embryonic cells from annual killifish to complement existing in vivo developmental studies has been hindered by a lack of efficient isolation techniques. Here, we present a protocol to isolate annual killifish blastoderm cells, at the epiboly and early dispersion phase, from embryos. We describe steps for hair removal, embryo cleaning, dechorionation, and cell purification. This protocol may also be used to develop strategies to isolate cells from embryos presenting similar challenges. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.