Trophoblastic cells play a crucial role in implantation and placentogenesis. A large proportion of the failures of conception in cows occur in the peri-implantation period, which are known as early embryo losses. In exploring this critical phenomenon, trophoblastic cell lines can provide substantial information. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stock in the long term. In this study, 12 new cell lines were established using bone morphogenetic protein 4 (BMP4). BMP4 stimulated embryonic cells to enter the trophoblastic cell lineage but there were no significant differences between intact and BMP4-treated groups. Only one out of 49 embryos developed trophoblastic cells in the intact group. Finally, 12 cell lines were maintained for around 30 passages, and they retained trophoblastic characteristics and expressed bovine trophoblastic genes: placental lactogen, interferon-τ, pregnancy-associated glycoprotein 1, and prolactin-related protein 1. Although the gene expression patterns were different among cell lines and depended on the cells, there was no significant relationship between the expression intensities of genes and the treatment dose of BMP4. All of them expressed bovine POU domain class 5 transcription factor 1 and caudal-type homeobox 2. The expression of these genes was confirmed by quantitative RT-PCR and immunohistochemical detection. These results suggest that BMP4 is involved in the raising of trophoblast cell lines from early embryonic cells and the newly developed cell lines can provide different types of bovine trophoblastic cells with different cell lineages. This may constitute a significant new tool for the examination of trophoblastic differentiation.