<span class="paragraphSection"><div class="boxTitle">Abstract</div>Embryonic genome activation (EGA) marks a critical developmental transition, yet its regulatory architecture remains incompletely defined. Here, we employed optimized low-input SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing) to map the temporal hierarchy of nascent transcription during mouse EGA. We uncovered patterns of transcriptional priming characterized by pre-activated genes (PAGs) with permissive chromatin states, followed by pronounced accumulation of PAGs-encoded proteins in blastocysts, suggesting that EGA memory propagates from early transcriptional activation to later lineage commitment. Furthermore, Integrative analysis nominated two-cell nascent transcription factors (TFs) as candidate regulators of the first lineage specification. Functional investigations demonstrated KLF17 as a key TF linking EGA to the first lineage specification via regulation of PAGs transcription. KLF17 deficiency led to the failure of transcriptional activation in approximately half of PAGs at the two-cell stage. Our work provides a detailed framework for decoding mammalian EGA and offers insights into how embryonic transcriptional priming is coordinated with early cell fate specification.</span>