Of Flemish descent, Flemming was born in Sachenberg, Germany, where his father worked as the director of an insane asylum. As a young man, Flemming showed great aptitude for literature and language, but chose instead to study medicine at several German universities. He served briefly as a hospital assistant before becoming an assistant in the department of zoology at Wurzburg University. In 1872, he accepted a position at the University of Prague, but the rise of Czech nationalism among his colleagues and students compelled him to return to Germany. He eventually became professor of anatomy and director of the Anatomical Institute at the University of Kiel, where he remained until his retirement.

Prior to Flemming's work, little headway had been made in the field of cytology since Matthias Schleiden and Theodor Schwann had proposed their cell theory in 1839, due in large part to the lack of effective cell staining...