After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos. The developmental transitions can be distinguished based on distinctive gene expression profiles, and we identified paired box 6 (PAX6) and aquaporin 3 (AQP3) expressed in early and late developmental stages, respectively. Two lineages can be segregated in porcine early and late blastocysts by the expression patterns of lineage-specific genes such as DAB2, clathrin adaptor protein (DAB2) for trophectoderm (TE), platelet derived growth factor receptor alpha (PDGFRA), Nanog homeobox (NANOG), fibronectin 1 (FN1), hepatocyte nuclear factor 4 alpha (HNF4A), goosecoid homeobox (GSC), nuclear receptor subfamily 5 group A member 2 (NR5A2), and lysine acetyltransferase 6A (KAT6A; previously known as MYST3) for inner cell mass (ICM). However, the epiblast and primitive endoderm cannot be identified in late blastocysts, and those TE or ICM lineage-specific genes were low expressed in blastomeres from the morula. Our results shed light on early cell fate determination in porcine preimplantation embryos and offer theoretical support for deriving porcine embryonic stem cells.