The event can be dated to 31 January 1632: the Amsterdam Guild of Surgeons, of which Tulp was official City Anatomist, permitted only one public dissection a year, and the body would have to be that of an executed criminal.

[2] Anatomy lessons were a social event in the 17th century, taking place in lecture rooms that were actual theatres, with students, colleagues and the general public being permitted to attend on payment of an entrance fee.

Rembrandt's image is a fiction; in a typical anatomy lesson, the surgeon would begin by opening the chest cavity and thorax because the internal organs there decay most rapidly.

Instead we see in the lower right corner an enormous open textbook on anatomy, possibly the 1543 De humani corporis fabrica (Fabric of the Human Body) by Andreas Vesalius.

The French art historian Jean-Marie Clarke suggests that the navel of the corpse has the shape of a capital R and connects this observation to the fact that Rembrandt worked intensively on his signatures in 1632, using three types consecutively before settling on the final, first name form in 1633.

In her 2014 historical fiction novel The Anatomy Lesson, author and journalist Nina Siegal tells the life story of Aris Kindt, based on documents about his criminal history that she discovered in the Amsterdam city archives.

Nelson Mandela was the cadaver, Nkosi Johnson was the instructor, and the students were Desmond Tutu, F. W. de Klerk, Thabo Mbeki, Jacob Zuma, Cyril Ramaphosa, Trevor Manuel, and Helen Zille.

The 2011 video game Deus Ex: Human Revolution references the painting in both in-game portraits that can be found on the wall and in a certain cinematic trailer, featuring the main protagonist Adam Jensen as the cadaver as Dr. Nicolaes and his students study his charred and ruined arms, which in the actual story become amputated and replaced with mechanical limbs.

In the 2012 German film Barbara, there is a scene in which a doctor offers his interpretation of the painting to a colleague (the protagonist) when she points out the inaccuracy of Aris Kindt's left hand.

2014's The Anatomy Lesson by Nina Siegal is a fictionalized account of the painting's creation and backstory, based on six years of historical research and archival documents about Aris Kindt's life.