Which of the following political factions challenged the validity of the Northwest Ordinance's prohibition of slavery by the late 1850s?

A.) Northern Democrats

B.) Southern Democrats

C.) Republicans

D.) Free-Soilers.

Southern Democrats is the correct answer.

It was the issue of slavery which mainly divided US into northern and southern states and further Democratic party into southern Democrats and Northern Democrats. The Southern Democrats were pro-slavery and thus challenged the Northwest Ordinance's prohibition of slavery by the late 1850s.